Optimized Multabody Constructs, Compositions, and Methods

Abstract

In an embodiment, the self-assembled polypeptide complex comprises: (a) a plurality of first fusion polypeptides, each of the first fusion polypeptides comprising (1) an Fc polypeptide and (2) an Fc polypeptide linked to a nanocage monomer or a subunit thereof, the Fc polypeptide comprising an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class; and (b) a plurality of second fusion polypeptides, each of the second fusion polypeptides comprising (1) an antigen-binding antibody fragment and (2) an antigen-binding antibody fragment linked to a nanocage monomer or a subunit thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 220,920 (filed July 12, 2021) and U.S. Provisional Patent Application No. 63 / 289,016 (filed December 13, 2021), the entire contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 12, 2022, is named "Sequence Listing Jul-2022 3206-5068.txt" and is 38,881 bytes in size. [Background technology]

[0003] Therapeutics based on antibodies or antibody fragments are being developed for a variety of uses, eg, to treat a variety of diseases or conditions.

[0004] However, antibody-based therapeutics may need to be tailored to have desirable characteristics (e.g., desirable biodistribution, half-life, etc.) after administration to a subject. Some antibody-based therapeutics have a format that differs from that of a natural immunoglobulin molecule. For example, in some therapeutics, the antibody or antibody fragment is fused to another polypeptide, and in some cases, the antibody or antibody fragment(s) is in a configuration or has a valency not found in nature. These antibody-based therapeutics may also need to be tailored.

[0005] Thus, there remains a need for optimized antibody-based therapeutics. Summary of the Invention

[0006] The present invention addresses this need by providing a suite of optimized self-assembling polypeptide complexes that include antibody fragments. Depending on the desired characteristics (e.g., pharmacokinetic characteristics), a particular self-assembling polypeptide complex or set of complexes can be selected for use. For example, in certain embodiments, self-assembling polypeptide complexes are provided that have half-lives or other pharmacokinetic characteristics similar to those of IgG molecules.

[0007] According to one aspect, (a) a plurality of first fusion polypeptides, each of which comprises (1) an Fc polypeptide linked to (2) a nanocage monomer or a subunit thereof, the Fc polypeptide comprising an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class; (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising: (1) an antigen-binding antibody fragment; and (2) a plurality of second fusion polypeptides comprising an antigen-binding antibody fragment linked to a nanocage monomer or a subunit thereof.

[0008] In one aspect, (1) if the Fc polypeptide is an IgG1 Fc polypeptide, the antigen-binding fragment is not a Fab fragment that binds to SARS-CoV-2, and / or (2) if the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds to CD19.

[0009] In one embodiment, the nanocage monomer is a ferritin monomer.

[0010] In some embodiments, the ferritin monomer is a ferritin light chain.

[0011] In some embodiments, the self-assembled polypeptide complex does not include any ferritin heavy chain or subunits of a ferritin heavy chain.

[0012] In some embodiments, the ferritin monomer is human ferritin.

[0013] In one embodiment, the Fc polypeptide is an IgG1 Fc polypeptide.

[0014] In one embodiment, the Fc polypeptide is an IgG2 Fc polypeptide.

[0015] In certain embodiments, the Fc polypeptide is a single chain Fc (scFc).

[0016] In certain embodiments, the Fc polypeptide is an Fc monomer.

[0017] In certain aspects, the antigen-binding antibody fragment comprises a light chain variable domain and a heavy chain variable domain.

[0018] In certain embodiments, the antigen-binding antibody fragment is a Fab fragment.

[0019] In certain embodiments, each second fusion polypeptide does not contain any CH2 or CH3 domains.

[0020] In certain embodiments, the one or more mutations comprise a mutation or set of mutations that are associated with altered binding to FcRn.

[0021] In one aspect, the mutation or set of mutations comprises mutations at one or more of the following residues: M252, I253, S254, T256, K288, M428, and N434 (numbering according to the EU index), or a combination thereof.

[0022] In one embodiment, the mutation or set of mutations comprises mutations at M428 and N434 (numbering according to the EU index).

[0023] In one embodiment, the mutation or set of mutations comprises the M428L and N434S mutations (numbering according to the EU index).

[0024] In one embodiment, the altered binding to FcRn is decreased binding to FcRn.

[0025] In certain aspects, the mutation or set of mutations associated with reduced binding to FcRn is selected from the group consisting of I253A, I253V, and K288A (numbering according to the EU index), and combinations thereof.

[0026] In certain embodiments, the one or more mutations include a mutation or set of mutations that are associated with altered effector function.

[0027] In one aspect, the Fc polypeptide is an IgG1 Fc polypeptide and the mutation or set of mutations comprises mutations at one or more of the following residues: L234, L235, G236, G237, P329, and A330 (numbering according to the EU index), or a combination thereof.

[0028] In one aspect, the altered effector function is a reduced effector function.

[0029] In one embodiment, the mutation or set of mutations associated with reduced effector function is selected from the group consisting of LALA (L234A / L235A), LALAP (L234A / L235A / P329G), G236R, G237A, A330L (numbering according to the EU index).

[0030] In one embodiment, the nanocage monomer or subunit thereof is a ferritin monomer subunit; a. each first fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, or b. Each first fusion polypeptide contains a ferritin monomer subunit that is an N-half ferritin, and each second fusion polypeptide contains a ferritin monomer subunit that is a C-half ferritin.

[0031] In one embodiment, the self-assembled polypeptide complex is characterized by a 1:1 ratio of the first fusion polypeptide to the second fusion polypeptide.

[0032] In certain embodiments, the Fc polypeptide is linked to a nanocage monomer or subunit thereof within each first fusion polypeptide via an amino acid linker.

[0033] In certain embodiments, the Fc polypeptide is linked to a nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof within each first fusion polypeptide.

[0034] In certain embodiments, the antigen-binding antibody fragment is linked to a nanocage monomer or subunit thereof within each second fusion polypeptide via an amino acid linker.

[0035] In certain embodiments, the antigen-binding antibody fragment is linked to a nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof within each second fusion polypeptide.

[0036] In one embodiment, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each of which comprises (1) an antigen-binding antibody fragment linked to (2) a nanocage monomer or a subunit thereof, and the third fusion polypeptide is different from the second fusion polypeptide.

[0037] In certain aspects, the antigen-binding antibody fragment in the third fusion polypeptide comprises a light chain variable domain and a heavy chain variable domain.

[0038] In certain embodiments, the antigen-binding antibody fragment in the third fusion polypeptide is a Fab fragment.

[0039] In certain embodiments, each third fusion polypeptide does not contain any CH2 or CH3 domains.

[0040] In one aspect, the antigen-binding antibody fragment of the second fusion polypeptide is capable of binding to a first epitope and the antigen-binding fragment of the third fusion polypeptide is capable of binding to a second epitope, wherein the first epitope and the second epitope are distinct and non-overlapping.

[0041] In one embodiment, the first epitope and the second epitope are from the same protein.

[0042] In one embodiment, the self-assembled polypeptide complex comprises a total of 24 to 48 fusion polypeptides.

[0043] In some embodiments, the self-assembled polypeptide complex comprises a total of at least 24 fusion polypeptides.

[0044] In some embodiments, the self-assembled polypeptide complex comprises a total of at least 32 fusion polypeptides.

[0045] In some embodiments, the self-assembled polypeptide complex has a total of about 32 fusion polypeptides.

[0046] In certain embodiments, the half-life of the self-assembling polypeptide complex when administered to a subject in need thereof is at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days.

[0047] In one aspect, the self-assembled polypeptide complex is characterized in that, after administration of a composition comprising the self-assembled polypeptide complex, the self-assembled polypeptide complex has a half-life substantially similar to the half-life of a reference IgG molecule administered by the same route of administration and in a similar composition.

[0048] In certain embodiments, the reference IgG molecule is the antibody from which the antigen-binding antibody fragment in the second fusion polypeptide is derived, or is the antibody from which the antigen-binding antibody fragment in the third fusion polypeptide is derived.

[0049] In one embodiment, the half-life of the self-assembling polypeptide complex is from about 3 to about 35 days when administered to a subject in need thereof.

[0050] In certain embodiments, the self-assembled polypeptide complex is detectable in serum at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration to a subject in need thereof.

[0051] In certain embodiments, the area under the curve (AUC) of the self-assembling polypeptide complex, when administered to a subject in need thereof, is at least 10 days·μg / mL, at least 25 days·μg / mL, at least 50 days·μg / mL, at least 100 days·μg / mL, at least 200 days·μg / mL, at least 300 days·μg / mL, at least 400 days·μg / mL, at least 500 days·μg / mL, at least 750 days·μg / mL, at least 1000 days·μg / mL, at least 1500 days·μg / mL, at least 2000 days·μg / mL, at least 2500 days·μg / mL, at least 3000 days·μg / mL, at least 4000 days·μg / mL, at least 5000 days·μg / mL, at least 6000 days·μg / mL, at least 7000 days·μg / mL, or at least 8000 days·μg / mL.

[0052] In one embodiment, the area under the curve (AUC) of the self-assembling polypeptide complex, when administered to a subject in need thereof, is about 10 to about 8000 day·μg / mL.

[0053] In one embodiment, the maximum concentration of the self-assembled polypeptide complex (C max), when administered to a subject in need thereof, is at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 250 μg / mL, at least 500 μg / mL, at least 750 μg / mL, at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, at least 100 mg / mL, at least 250 mg / mL, at least 500 mg / mL, or at least 750 mg / mL.

[0054] In one embodiment, the maximum concentration of the self-assembled polypeptide complex (C max ) is from about 10 μg / mL to about 750 mg / mL when administered to a subject in need thereof.

[0055] In some embodiments, the subject is a human.

[0056] In some embodiments, administration to a subject is by parenteral administration.

[0057] In certain embodiments, administration to the subject is subcutaneous, intravenous, intramuscular, intranasal, or by inhalation.

[0058] In one aspect, the self-assembled polypeptide complex is characterized as inducing antibody-dependent cellular phagocytosis (ADCP) in an in vitro model of ADCP.

[0059] In certain embodiments, the ADCP is induced at a level of internalization of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the target.

[0060] According to certain aspects, methods are provided that include administering to a mammalian subject a composition that includes a self-assembled polypeptide complex as described herein.

[0061] In some embodiments, the subject is a human.

[0062] In some embodiments, the method comprises administration by a systemic route.

[0063] In some embodiments, systemic routes include subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

[0064] In certain embodiments, the half-life of the self-assembled polypeptide complex in a mammalian subject following administration is at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days.

[0065] In some embodiments, the half-life of the self-assembling polypeptide complex in a mammalian subject following administration is between 3 and 35 days.

[0066] In certain embodiments, the area under the curve (AUC) of the self-assembling polypeptide complex in the mammalian subject following administration is at least 10 days·μg / mL, at least 25 days·μg / mL, at least 50 days·μg / mL, at least 100 days·μg / mL, at least 200 days·μg / mL, at least 300 days·μg / mL, at least 400 days·μg / mL, at least 500 days·μg / mL, at least 750 days·μg / mL, at least 1000 days·μg / mL, at least 1500 days·μg / mL, at least 2000 days·μg / mL, at least 2500 days·μg / mL, at least 3000 days·μg / mL, at least 4000 days·μg / mL, at least 5000 days·μg / mL, at least 6000 days·μg / mL, at least 7000 days·μg / mL, or at least 8000 days·μg / mL.

[0067] In one embodiment, the area under the curve (AUC) of the self-assembling polypeptide complex in a mammalian subject following administration is from about 10 to about 8000 day·μg / mL.

[0068] In certain embodiments, the maximum concentration (Cmax) of the self-assembling polypeptide complex in the mammalian subject following administration is at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 250 μg / mL, at least 500 μg / mL, at least 750 μg / mL, at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, at least 100 mg / mL, at least 250 mg / mL, at least 500 mg / mL, or at least 750 mg / mL.

[0069] In one embodiment, the maximum concentration (Cmax) of the self-assembling polypeptide complex in a mammalian subject following administration is from about 10 μg / mL to about 750 mg / mL.

[0070] According to one aspect, a fusion polypeptide is provided, comprising: (1) a fusion polypeptide comprising: (2) an Fc polypeptide linked to a nanocage monomer or a subunit thereof, wherein the Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, wherein the one or more mutations comprise a mutation or set of mutations associated with altered binding to FcRn and / or altered effector function.

[0071] In one embodiment, the nanocage monomer is a ferritin monomer.

[0072] In one embodiment, the ferritin monomer is a ferritin light chain.

[0073] In some aspects, the fusion polypeptide does not contain any ferritin heavy chain or subunits of a ferritin heavy chain.

[0074] In some embodiments, the ferritin monomer is human ferritin.

[0075] In one embodiment, the Fc polypeptide is an IgG1 Fc polypeptide.

[0076] In one embodiment, the Fc polypeptide is an IgG2 Fc polypeptide.

[0077] In certain embodiments, the Fc polypeptide is a single chain Fc (scFc).

[0078] In one aspect, the mutation or set of mutations comprises mutations at one or more of the following residues: M252, I253, S254, T256, K288, M428, and N434 (numbering according to the EU index), or a combination thereof.

[0079] In one embodiment, the altered binding to FcRn is decreased binding to FcRn.

[0080] In certain aspects, the mutation or set of mutations associated with reduced binding to FcRn is selected from the group consisting of I253A, I253V, and K288A (numbering according to the EU index), and combinations thereof.

[0081] In one aspect, the Fc polypeptide is an IgG1 Fc polypeptide and the mutation or set of mutations comprises mutations at one or more of the following residues: L234, L235, G236, G237, P329, and A330 (numbering according to the EU index), or a combination thereof.

[0082] In one aspect, the altered effector function is a reduced effector function.

[0083] In one embodiment, the mutation or set of mutations associated with reduced effector function is selected from the group consisting of LALA (L234A / L235A), LALAP (L234A / L235A / P329G), G236R, G237A, A330L (numbering according to the EU index).

[0084] In one embodiment, the nanocage monomer or subunit thereof is a ferritin monomer subunit; a. each first fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, or b. Each first fusion polypeptide contains a ferritin monomer subunit that is an N-half ferritin, and each second fusion polypeptide contains a ferritin monomer subunit that is a C-half ferritin.

[0085] In certain embodiments, the Fc polypeptide is linked to a nanocage monomer or subunit thereof within each first fusion polypeptide via an amino acid linker.

[0086] In certain embodiments, the Fc polypeptide is linked to a nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof within each first fusion polypeptide. [Brief description of the drawings]

[0087] [Figure 1A] FIG. 1 is a diagrammatic representation of human ferritin light chain (hFTL) and exemplary N-half ferritin and C-half ferritin molecules.

[0088] [Figure 1B] 1 is a diagrammatic representation of an exemplary Multabody formation of the present disclosure. [Figure 1C] 1 is a diagrammatic representation of an exemplary Multabody formation of the present disclosure. [Figure 1D] 1 is a diagrammatic representation of an exemplary Multabody formation of the present disclosure.

[0089] [Figure 2A] Negative stain electron micrographs of T-01 MB, T-02 MB, and T-01 MB.v2, each containing wild-type IgG1 Fc.

[0090] [Figure 2B] 1 shows negative staining electron micrographs of T-01 MB containing various Fc.

[0091] [Figure 3A] 13 provides Biolayer Interferometry (BLI) concentration response curves for binding of T-01 MB containing various Fc to human FcRn at pH 5.6 and pH 7.4, respectively. [Figure 3B] 13 provides Biolayer Interferometry (BLI) concentration response curves for binding of T-01 MB containing various Fc to human FcRn at pH 5.6 and pH 7.4, respectively.

[0092] [Figure 3C] 1 provides BLI concentration response curves for binding of various Fc-containing T-01 MBs to human FcγRIIa, human FcγRIIb, and FcγRI, respectively. [Figure 3D] 1 provides BLI concentration response curves for binding of various Fc-containing T-01 MBs to human FcγRIIa, human FcγRIIb, and FcγRI, respectively. [Figure 3E] 1 provides BLI concentration response curves for binding of various Fc-containing T-01 MBs to human FcγRIIa, human FcγRIIb, and FcγRI, respectively.

[0093] [Figure 3F] 1 provides BLI concentration response curves for binding of T-01 MB.v2 containing wild type IgG1 Fc or Fc IgG1 with M428L / N434S (LS) mutations to human Fc receptors.

[0094] [Figure 4A] 13 provides BLI concentration response curves for binding of T-01 MB, T-02 MB, and T-01 MB.v2 to the target / epitope of PGDM1400, N49P7, 10E8v4, or iMab included as the Fab of the Multabody.

[0095] [Figure 4B]FIG. 13 provides BLI concentration response curves for binding of T-01 MB containing various Fc to targets / epitopes of PGDM1400 (BG5050 SOSIP.664 D368R), N49P7 (93TH057), and 10E8v4 (gp41 MPER), respectively. [Figure 4C] FIG. 13 provides BLI concentration response curves for binding of T-01 MB containing various Fc to targets / epitopes of PGDM1400 (BG5050 SOSIP.664 D368R), N49P7 (93TH057), and 10E8v4 (gp41 MPER), respectively. [Figure 4D] FIG. 13 provides BLI concentration response curves for binding of T-01 MB containing various Fc to targets / epitopes of PGDM1400 (BG5050 SOSIP.664 D368R), N49P7 (93TH057), and 10E8v4 (gp41 MPER), respectively.

[0096] [Figure 4E] 1 provides BLI concentration response curves for binding of PGDM1400, N49P7, 10E8v4, or iMab IgG to BG5050 SOSIP.664 D368R, 93TH057, gp41 MPER, and CD4.

[0097] [Figure 5A] 1 shows experiments performed using CB17 / Icr-Prkdcscid / IcrIcoCrl immunodeficient (SCID) mice, as described in Example 4. [Figure 5B] Figures 5B, 5C, 5D, and 5E show serum levels of test Multabody or IgG1 control following subcutaneous administration in SCID mice. [Figure 5C] Figures 5B, 5C, 5D, and 5E show serum levels of test Multabody or IgG1 control following subcutaneous administration in SCID mice. [Figure 5D] Figures 5B, 5C, 5D, and 5E show serum levels of test Multabody or IgG1 control following subcutaneous administration in SCID mice. [Figure 5E] Figures 5B, 5C, 5D, and 5E show serum levels of test Multabody or IgG1 control following subcutaneous administration in SCID mice.

[0098] [Figure 6A] 1 shows experiments performed using NOD / Shi-scid / IL-2Rγnull immunodeficient (NCG) mice, as described in Example 4. [Figure 6B] FIG. 6B shows serum levels of test Multabody or IgG1 control following subcutaneous administration in NCG mice. [Figure 6C] Figure 6C provides the body weight of NCG mice upon administration of test Multabody or IgG1 control. Mean ± SD of n=3 mice is shown.

[0099] [Figure 7] Figure 1 shows dose-dependent phagocytosis induced by test multibody or control, quantified as the percentage increase in internalization of 93TH057-coated microspheres compared to uncoated microspheres. *P<0.05, ***P<0.001, and P****<0.0001. n=4 biologically independent samples.

[0100] [Figure 8A] Shown are the breadth and median IC50 values ​​(μg / mL) of the test multibodies (T-01 MB, T-01 MB.v2, and T-02 MB., respectively) (diamonds), parental antibodies PGDM1400, N49P7, and 10E8v4 (circles), IgG1 control (triangles), and the N6 / PGDM1400×10E8v4 trispecific antibody (inverted triangles), as determined by the TZM-bl assay described in Example 6. [Figure 8B]Shown are the breadth and median IC50 values ​​(μg / mL) of the test multibodies (T-01 MB, T-01 MB.v2, and T-02 MB., respectively) (diamonds), parental antibodies PGDM1400, N49P7, and 10E8v4 (circles), IgG1 control (triangles), and the N6 / PGDM1400×10E8v4 trispecific antibody (inverted triangles), as determined by the TZM-bl assay described in Example 6. [Figure 8C] Shown are the breadth and median IC50 values ​​(μg / mL) of the test multibodies (T-01 MB, T-01 MB.v2, and T-02 MB., respectively) (diamonds), parental antibodies PGDM1400, N49P7, and 10E8v4 (circles), IgG1 control (triangles), and the N6 / PGDM1400×10E8v4 trispecific antibody (inverted triangles), as determined by the TZM-bl assay described in Example 6.

[0101] [Figure 8D] 1 shows dose-dependent neutralization of HIV-1 by T-01 MB containing various Fc's as determined by the TZM-bl assay described in Example 6.

[0102] [Figure 9A] 1 illustrates the inhibition of CXCR4-tropic HIV-1 isolate IIIB infection of PBMCs by T-01 MB, T-01 MB.v2, or IgG1 control. Mean values ​​± SD for three technical replicates are shown. [Figure 9B] FIG. 9B shows the percentage of viable cells following T-01 MB, T-01 MB.v2, or IgG1 control relative to untreated control cells.

[0103] [Figure 10] 10A and 10B show the stability of T-01 MB and T-01 MB.v2 under temperature stress conditions (40° C.) for 4 weeks. See Example 8.

[0104] [Figure 11]HIV-1 bNAb multimerization increases neutralization potency. (A) Schematic of self-assembly of apoferritin (24 subunits) and (B) single-chain Fab-apoferritin fusions. The Fab light chain (LC) and heavy chain (HC) are shown in light and dark pink, respectively, and are connected to the N-terminus of the light chain of human apoferritin (gray) via a GGS-like flexible linker (dark). (C) Schematic of different Fab densities displayed on human apoferritin. Co-transfection of plasmids encoding scFab-human apoferritin with unconjugated apoferritin in ratios of 1:4 (dark yellow), 1:1 (black), 4:1 (blue), and 1:0 (red) resulted in molecules with different scFab titers, as confirmed by elution volumes in size-exclusion chromatography and less unconjugated apoferritin in SDS-PAGE. Negative staining electron micrographs of samples with the lowest and highest scFab valence are shown (scale bar 50 nm). (D) Avidity effect on neutralization of five bNAbs against a panel of five PsVs (PVO.04, JRCSF, BG505 T332N, THRO4156.18 and t278-50). Fold potency increase was calculated by dividing parental IgG IC50 (μg / mL) by Fab-apoferritin fusion IC50 (μg / mL). Fold potency increase analysis was omitted in the following cases: N49P7-t278-50, VRC01-T278-50 and 10-1074-THRO4156.18 due to neutralization resistance. Bars (±SD) represent the mean from n=3 biologically independent samples.

[0105] [Figure 12]Characterization of scFab-apoferritin fusions. (A) SDS-PAGE bands corresponding to scFab-apoferritin and unconjugated apoferritin were quantified by densitometry using ImageJ software (rsb.info.nih.gov / ij / ). Intensity plots of the bands in each lane (yellow boxes) are shown (B). (C) The approximate number of scFabs displayed on the particles was calculated as follows: intensity of scFab band / total intensity, and compared with the theoretical number estimated from the DNA ratio used for cotransfection.

[0106] [Figure 13] Design, assembly, and neutralization profile of HIV Multabody against 14-PsV panel. (A) Schematic of human apoferritin split design driving scFab-human apoferritin subunit heterodimerization. (B) In-line size-exclusion chromatography of 24-mer PGDM1400 scFab-apoferritin particles (black) and T-01 MB (dark red) followed by multi-angle light scattering. The molar mass of each elution peak (lines under UV absorbance) is shown in MDa. (C) Negative stain electron micrograph of T-01 MB (scale bar 50 nm). (D) Concentration-response curves for binding of T-01 MB to multiple epitopes. PGDM1400, N49P7, and 10E8 binding sites are colored red, blue, and pink, respectively, on the surface representation of HIV-1 Env trimer (grey). Red lines represent raw data and black lines represent global fits. (E) Range (cutoff IC50 set at 10 μg / mL) and median IC50 values ​​(μg / mL) of T-01 MB (red diamonds), parental bNAb (white circles), IgG combination (grey triangles), and N6 / PGDM1400x10E8v4 trispecific antibody (black triangles). The 14-PsV panel was selected based on susceptibility and resistance to parental IgG. (F) Individual IC50 values ​​(μg / mL) for each PsV variant. The solid line indicates the median neutralization IC50 of all 14 virus strains. The pseudovirus showing the highest neutralization resistance is highlighted with a red box. IC50 values ​​in (D) and (E) were calculated from three biological replicates.

[0107] [Figure 14] Multabody affinity purification scheme. Sequential affinity purification of Protein A and Protein L. Binding to Protein A enriches Multabodies with Fc (green), whereas Protein L enriches Multabodies with kappa chain Fab PGDM1400 (blue). Complementarity of the two halves of the apoferritin split design ensures the presence of N49P7 / iMab (orange) and 10E8v4 scFabs (pink) (fused to C-ferritin) during the Protein A purification step. An alanine to proline point mutation at position 12 of the kappa chain of iMab was introduced to disrupt binding to Protein L. Gel filtration is performed to separate any aggregated material or disorganized components.

[0108] [Figure 15] Generation of Multabodies Cross-targeting HIV-1 Env and CD4 Receptors. (A) Schematic of MB components. (B) Size-exclusion chromatography along with multi-angle light scattering of 24-mer PGDM1400 scFab-apoferritin particles (black) and T-02 MB (blue). Molar mass of each sample is shown in MDa. (C) Negative stain electron micrographs (scale bar 50 nm) and (D) binding profile of T-02 MB. (E) Width (cutoff IC50 set at 10 μg / mL) and median IC50 values ​​(μg / mL) of T-02 MB (red diamonds) compared to parental bNAb (white circles) and IgG combinations (grey triangles). Mean IC50 values ​​(μg / mL) derived from three biological replicates for those pseudoviruses showing the highest neutralization resistance to T-02 MB are shown.

[0109] [Figure 16] Biophysical characterization of HIV-1 multibodies. Comparison of Tm and Tagg temperatures of T-01 / T-02 MB, 12-mer ferritin fusion, parental IgG, and N6 / PGDM1400x10E8v4 trispecific antibody.

[0110] [Figure 17] Binding characteristics of IgG binding to four different antigens. BLI response curves of IgG binding to 93TH057 gp120, BG505 SOSIP.664_D368R, MPER peptide and CD4 immobilized on Ni-NTA biosensor. BG505 SOSIP.664_D368R trimer and 93TH057 gp120 monomer were selected as epitope-specific ligands for PGDM1400 and N49P7, respectively. Red lines represent raw data and black lines represent global fits.

[0111] [Figure 18] Engineering and biophysical characterization of Multabodyv2. (A) The second generation Multabody design shows two distinct features compared to the original Multabody design: 1) Fc (green) is fused to the C-terminus of the second half of apoferritin in a split-ferritin design, and 2) a single chain Fc domain (green) fused to the C-terminus of the apoferritin half protomer reverts to a monomeric Fc chain. Dimerization of each Fc in MB.v2 drives the organization of four Fabs (two Fab2 and two Fab3, bottom row), whereas only one Fab per Fc is incorporated in the previous MB version (top row). (B) Negative stain electron micrographs of T-01 MB.v2 (scale bar 50 nm). (C) Concentration-response curves for binding of T-01 MB.v2 to multiple epitopes. Red lines represent raw data, black lines represent global fits. Comparison of the long-term stability of (D) Tagg and (E) two different Multabody versions under temperature stress conditions (10 mg / ml, 40 °C). Comparison of PsV neutralization (mean ± SD of two technical replicates) at weeks 0 and 4 is shown.

[0112] [Figure 19]Characterization of Multabody v2. (A) Modification of the fusion of Fc (green) from the N-terminus of N-ferritin (top) to the C-terminus of C-ferritin (bottom) inverts the orientation of Fc within the Multabody. (B) Fc dimerization of two Fc chains fused to the C-terminus of two independent ferritin subunits at the 4-fold symmetry axis of the apoferritin nanocage serves as an additional driver of Multabody v2 assembly.

[0113] [Figure 20] Fine-tuning the Fc on the Multabody for IgG-like properties. (A) Concentration response curves of pH-dependent binding to human FcRn by T-01 MB and T-01 MB.v2. (B) Comparison of apparent binding affinity (KD) of FcRn at acidic pH between MB and IgG1. n=3 biologically independent samples are shown. Apparent KD below 10-12M (dashed black line) is above the detection limit of the instrument. (C) Concentration response curves for high affinity human FcγRI (top) and low affinity human FcγRIIa (bottom). (D) Dose-dependent phagocytosis determined as the increase in the percentage of internalization of fluorescent microspheres coated with 93TH057 compared to no antibody control. Anti-human FcR binding inhibitor antibody was added to block Fc-mediated internalization (dark red). Data were analyzed by two-way ANOVA with Tukey's multiple comparison test. Each group was compared to the IgG negative control. *P<0.05, ***P<0.001, and P****<0.0001. IgG and MB samples with no affinity for antigen-coated beads were added as control samples. n=4 biologically independent samples. (E) Serum levels after subcutaneous administration of 5 mg / kg Multabody or parental IgG mixture in female NOD / Shi-scid / IL-2Rγnull (NCG) immunodeficient mice. (F) Body weight at the time of administration of 5 mg / kg of molecules to NCG mice. Mean ± SD for n=3 mice is shown in (E) and (F).

[0114] [Figure 21]Broad and potent neutralization by Multabody v2 against an extended HIV-1 PsV panel. (A) Range and median IC50 values ​​(μg / mL) of T-01 Multabody versions (different shades of red diamonds), individual IgGs (black circles) and IgG mixtures (blue triangles) against a 25-PsV panel with 56% of PsV mutants resistant to PGDM1400 neutralization. B) Individual IC50 values ​​(μg / mL) per PsV mutant. IC50 values ​​in (A) and (B) were calculated from three biological replicates. (C) Potency (IC50) range (left) and potency (IC80) range (right) curve comparison of T-01 Multabody versions and parental IgG and IgG mixtures against an extended multiclade panel of 118 HIV-1 PsV mutants. (D) Individual IC50 (left) and IC80 (right) values ​​for each PsV mutant in (C). Yellow dots correspond to IC50 values ​​from PsVs that are highly resistant to neutralization by PGDM1400. The solid lines in (B) and (D) indicate the median IC50 neutralization titers of all virus strains in each panel.

[0115] [Figure 22] Inhibition of PsV neutralization and primary PBMC infection by Multabodies. (A) Molar potency (IC50)-breadth (left) and molar potency (IC80)-breadth (right) graphs comparing the T-01 Multabody version and the parental IgG and IgG mixture against a panel of 118 HIV-1 PsV mutants. (B) Measurement of HIV-1 replication by p24 detection in PBMC culture supernatants derived from three different blood donors. Data shown represent HIV-1 replication levels 7 days after infection with the CXCR4-tropic HIV-1 isolate IIIB. Mean values ​​± SD of three technical replicates are shown. (C) Effect of IgG mixture and Multabody treatment on cell viability, as percentage of viable cells relative to untreated control cells, under the same experimental conditions as in (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] The inventors have previously described self-assembling polypeptide complexes that contain fusion polypeptides that contain antibody fragments. These self-assembling polypeptide complexes can be designed and adapted for various therapeutic purposes. For example, as described herein, self-assembling polypeptide complexes that contain both Fab and Fc-containing fusion polypeptides can be used to target cells that express antigens that Fab can bind, and the Fc portion can mediate interactions with other molecules in the body.

[0117] In many situations, it may be desirable to optimize the behavior of these self-assembled polypeptide complexes after administration, for example, by adjusting properties such as half-life and / or ability to mediate antibody-mediated effects. Techniques for optimizing IgG molecules are known in the art. For example, both half-life and specific antibody-mediated effects can be adjusted using modifications to the Fc region of IgG molecules.

[0118] However, the inventors have discovered that in some circumstances, techniques for optimizing IgG molecules can have surprising and unexpected effects when applied to our self-assembling polypeptide complexes. As an example, the inventors have discovered that Fc modifications typically associated with reduced FcRn binding (and reduced half-life) in the context of IgG1 molecules actually confer more desirable bioavailability properties in the context of our self-assembling polypeptide complexes.

[0119] Taking advantage of these and other insights, the inventors have developed a series of self-assembling polypeptide complexes and related methods, each optimized for a particular desired outcome.

[0120] For example, in certain embodiments, after administration to a subject, the self-assembling polypeptide complex provided has one or more pharmacokinetic characteristics similar to that of a reference IgG molecule (e.g., an IgG molecule whose class matches the class of the Fc chain in the Fc polypeptide in the self-assembling polypeptide complex). For example, in some embodiments, the self-assembling polypeptide complex disclosed herein has a bioavailability similar to that of the reference IgG molecule. In some embodiments, the self-assembling polypeptide complex disclosed herein has a half-life similar to that of the reference IgG molecule.

[0121] In certain embodiments, following administration to a subject, the self-assembled polypeptide complexes provided induce antibody-dependent cellular phagocytosis (ADCP).

[0122] definition The terms "about" and "approximately", when used herein in reference to a value, are used interchangeably and refer to a value similar to the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the relevant degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the terms "about" and "approximately" can encompass values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0123] As used herein, the terms "modify," "modified," "reduce," "reduced," "increase," "increased," or "reduction," "reduced" (e.g., with respect to a particular outcome or effect) have a meaning relative to a reference level. In some embodiments, in the context of considering a mutation in an Fc chain or Fc polypeptide, the reference level is a level known or determined in an IgG that does not contain the referenced mutation(s) in the Fc region.

[0124] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to a polypeptide (e.g., a ferritin chain) that can be assembled into a ferritin complex, typically comprising 24 protein subunits. In some embodiments, the ferritin is a human ferritin, e.g., a human ferritin light chain, e.g., a human ferritin light chain having at least 85% sequence identity with SEQ ID NO: 1 or UniProt P02792. In some embodiments, the ferritin is a wild-type ferritin. For example, the ferritin may be a wild-type human ferritin.

[0125] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that can self-assemble, in the presence of other ferritin chains, into a polypeptide complex containing multiple ferritin chains, for example, 24 or more ferritin chains.

[0126] As used herein, the term "linker" is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, one of skill in the art will understand that polypeptides having structures that include two or more functional or organizational domains (e.g., fusion polypeptides) often include a stretch of amino acids between such domains that link them together. In some embodiments, polypeptides that include linker elements have an overall structure of the general form S1-L-S2, where S1 and S2, which may be the same or different, represent two domains that are associated with each other by a linker (L). In some embodiments, the linker comprises an "amino acid linker" i.e., amino acid residues, e.g., the amino acid linker can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acid residues. In some embodiments, the linker is characterized in that it does not tend to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

[0127] The term "multispecificity" as used herein refers to the characteristic of having at least two binding sites to which at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors), can bind. For example, a polypeptide complex comprising at least two Fab fragments, each of which can bind a different antigen, is "multispecific". As an additional example, a polypeptide complex comprising an Fc fragment (capable of binding to an Fc receptor) and a Fab fragment (capable of binding to an antigen) is "multispecific".

[0128] As used herein, the term "multivalent" refers to the characteristic of having at least two binding sites to which a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor), can bind. The binding partners capable of binding to the at least two binding sites can be the same or different.

[0129] The term "nanocage monomer" as used herein refers to a single chain of a polypeptide that can self-assemble with other nanocage monomers to form a self-assembled polypeptide complex comprising a plurality of nanocage monomers. In some embodiments, the nanocage monomer is selected from monomers of ferritin, apoferritin, encapsulin, sulfur oxygenase reductase (SOR), lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P coat protein, MS2 coat protein, fragments thereof, and mutants thereof.

[0130] The term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three amino acids, e.g., a polymer linked together by peptide bonds. Those of skill in the art will appreciate that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Moreover, those of skill in the art will appreciate that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares an overall sequence identity with another polypeptide of the same class that is at least about 30-40%, often more than about 50%, about 60%, about 70%, or about 80%, and more usually more than 90% or even more than 95%, 96%, 97%, 98%, or 99%, usually encompassing at least 3-4, and often up to 20 or more amino acids, is encompassed by the related term "polypeptide" as used herein. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0131] The term "self-assembly," when used in reference to a macromolecular complex (e.g., a polypeptide complex), refers to the spontaneous formation of the complex when sufficient components of the complex to be formed (e.g., a fusion polypeptide) are present. In some embodiments, the complex self-assembles at physiological conditions or in a buffer (e.g., a solution) that corresponds to physiological conditions.

[0132] As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with or susceptible to the relevant disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics characteristic of susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is a subject to whom and / or to whom diagnosis and / or treatment is being administered.

[0133] As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of any therapy that partially or completely alleviates, alleviates, relieves, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of subjects who do not show signs of the associated disease, disorder, and / or condition, and / or who show only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of subjects who show one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0134] A. Fusion Polypeptides In many embodiments, a fusion polypeptide compatible with the compositions and methods disclosed herein generally comprises a nanocage monomer or subunit thereof linked to either an Fc polypeptide or an antigen-binding antibody fragment. Within the fusion polypeptide, the Fc polypeptide or antigen-binding antibody fragment may be linked to the nanocage monomer or subunit thereof at a particular terminus, e.g., the N-terminus or C-terminus, of the nanocage monomer or subunit thereof. In some embodiments, the Fc polypeptide or antigen-binding antibody fragment is linked via an amino acid linker, such as the linkers described herein. In some embodiments, (1) if the Fc polypeptide is an IgG1 Fc polypeptide, the antigen-binding fragment is not a Fab fragment that binds SARS-CoV-2, and / or (2) if the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds CD19.

[0135] 1. Nanocage monomer and its subunits In some embodiments, the nanocage monomer is a ferritin monomer.

[0136] The term "ferritin monomer" is used herein to refer to a single chain of ferritin that can self-assemble into a polypeptide complex containing multiple ferritin chains, for example 24 or more ferritin chains, in the presence of other ferritin chains. In some embodiments, the ferritin monomer is a ferritin light chain. In some embodiments, the ferritin monomer does not contain a ferritin heavy chain or other ferritin components that can bind iron.

[0137] In some embodiments, each fusion polypeptide in the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise any ferritin heavy chain or a subunit of a ferritin heavy chain.

[0138] In some embodiments, the ferritin monomer is a human ferritin chain, e.g., a human ferritin light chain, e.g., a human ferritin light chain having a sequence of at least residues 2-175 of SEQ ID NO: 1. In some embodiments, the ferritin monomer is a mouse ferritin chain.

[0139] A "subunit" of a ferritin monomer refers to a portion of a ferritin monomer that can spontaneously associate with another, separate subunit of a ferritin monomer such that the subunits together form a ferritin monomer, which can then self-assemble with other ferritin monomers to form a polypeptide complex.

[0140] In some embodiments, the ferritin monomer subunit comprises about half of a ferritin monomer. As used herein, the term "N-half ferritin" refers to about half of a ferritin chain that includes the N-terminus of the ferritin chain. As used herein, the term "C-half ferritin" refers to about half of a ferritin chain that includes the C-terminus of the ferritin chain. The exact point at which the ferritin chain may be split to form N-half ferritin and C-half ferritin may vary depending on the embodiment. For example, in the context of a ferritin monomer subunit based on a human ferritin light chain, the half may be split at a point corresponding to positions from about 75 to about 100 (or a substantial portion thereof) of SEQ ID NO:1. For example, in some embodiments, an N-half ferritin based on a human ferritin light chain has an amino acid sequence corresponding to residues 1-95 (or a substantial portion thereof) of SEQ ID NO:1, and a C-half ferritin based on a human ferritin light chain has an amino acid sequence corresponding to residues 96-175 (or a substantial portion thereof) of SEQ ID NO:1.

[0141] In some embodiments, each half is split at a point corresponding to positions from about 85 to about 92 of SEQ ID NO: 1. For example, in some embodiments, an N-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-90 of SEQ ID NO: 1, and a C-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 91-175 of SEQ ID NO: 1.

[0142] 2. Fc Polypeptides In certain embodiments, the fragment crystallizable (Fc) polypeptides each comprise an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, which may be, for example, of the IgG1 or IgG2 class, as described further below.

[0143] Unless otherwise stated, numbering of mutations within antibody fragments, e.g., Fc polypeptides, throughout this disclosure is according to the EU index.

[0144] In some embodiments, the Fc polypeptide is a human IgG Fc polypeptide, i.e., except for the mutations described herein, the Fc polypeptide comprises an Fc chain substantially similar to that of the Fc chain in wild-type human IgG.

[0145] In some embodiments, the Fc polypeptide is an IgG1 Fc polypeptide (e.g., a human IgG1 Fc polypeptide), i.e., except for the mutations described herein, the Fc polypeptide comprises an Fc chain having an amino acid sequence substantially similar to the sequence of the chain in wild-type IgG1 Fc. In some embodiments, the wild-type IgG1 Fc is a human IgG1 Fc, and each Fc chain has the amino acid sequence of SEQ ID NO:5.

[0146] For example, an IgG1 Fc polypeptide may comprise an Fc chain having an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the Fc chain in wild-type IgG1 Fc. In some embodiments, the IgG1 Fc polypeptide comprises an Fc chain having an amino acid sequence that includes the Fc mutations specifically described for that IgG1 Fc polypeptide, but is otherwise 100% identical to the Fc chain in wild-type IgG1 Fc. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO:5 by at least one, at least two, at least three, or at least four amino acid residues. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO:5 by no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 4 amino acid residues.

[0147] In some embodiments, the Fc polypeptide is an IgG2 Fc polypeptide (e.g., a human IgG2 Fc polypeptide), i.e., except for the mutations described herein, the Fc polypeptide comprises an Fc chain having an amino acid sequence substantially similar to the sequence of the chain in a wild-type IgG2 Fc. In some embodiments, the wild-type IgG2 Fc is a human IgG2 Fc, and each Fc chain has the amino acid sequence of SEQ ID NO:46.

[0148] For example, an IgG2 Fc polypeptide may comprise an Fc chain having an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the Fc chain in wild-type IgG2a Fc. In some embodiments, an IgG2 Fc polypeptide comprises an Fc chain having an amino acid sequence that includes the Fc mutations specifically described for that IgG2 Fc polypeptide, but is otherwise 100% identical to the Fc chain in wild-type IgG2 Fc. In some embodiments, an Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO:46 by at least one, at least two, at least three, or at least four amino acid residues. In some embodiments, an Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO:46 by no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 4 amino acid residues.

[0149] In some embodiments, the Fc polypeptide is a single chain Fc (scFc) that comprises two Fc chains linked together by a covalent linker, e.g., via an amino acid linker.

[0150] In some embodiments, the Fc polypeptide is an Fc monomer, e.g., a single Fc chain having only one CH2 domain (the second constant Ig domain of the heavy chain) and one CH3 domain (the third constant Ig domain of the heavy chain), which single Fc chain can typically dimerize with another single Fc chain.

[0151] In some embodiments, the one or more mutations include a mutation or set of mutations associated with an altered characteristic as further described herein. By "associated with" it is meant that the mutation or set of mutations has been previously characterized as conferring an altered characteristic (e.g., altered binding to FcRn, altered effector function, etc.) in the context of an antibody, such as an IgG antibody. By "altered" it is meant that the property (e.g., binding to an Fc receptor (e.g., FcRn)) is different from that observed without the mutation or set of mutations.

[0152] For example, in some embodiments, the altered characteristics include altered binding to an Fc receptor.

[0153] In some embodiments, the altered characteristics include altered binding to FcRn.

[0154] For example, a mutation or set of mutations associated with altered binding to FcRn may include mutations at one or more residues selected from the following: M252, I253, S254, T256, K288, M428, N434, or a combination thereof.

[0155] In some embodiments, altered Fc receptor binding comprises decreased binding to FcRn (e.g., decreased binding compared to a reference level corresponding to the level observed without the one or more mutations). For example, in some embodiments, the one or more mutations comprise a mutation or set of mutations associated with decreased binding to FcRn, e.g., I253A, I253V, K288A, or a combination thereof.

[0156] In some embodiments, the one or more mutations include a mutation or set of mutations associated with altered effector function, e.g., altered binding to an Fc receptor associated with effector function (e.g., an Fcγ receptor such as FcγRI, FcγRII, or FcγRIIb).

[0157] For example, the one or more mutations may include a mutation or set of mutations at one or more residues selected from the following: L234, L235, G236, G237, P329, A330, and combinations thereof.

[0158] In some embodiments, the altered binding to an Fc receptor comprises reduced effector function, for example, LALA (L234A / L235A), LALAP (L234A / L235A / P329G), G236R, G237A, A330L, or a combination thereof.

[0159] 3. Antigen-binding antibody fragment In certain embodiments, the antigen-binding antibody fragment comprises a heavy chain variable region (e.g., V H In certain embodiments, the antigen-binding antibody fragment comprises a heavy chain variable domain (e.g., V H ) and a light chain variable domain (e.g., V L or V K In certain embodiments, the antigen-binding antibody fragment comprises a heavy chain variable domain (e.g., V H ) and a light chain variable domain (e.g., V L or V K ) containing Fab.

[0160] In certain embodiments, the antigen-binding antibody fragment does not include any domains derived from the Fc region, e.g., does not include any CH2 or CH3 domains.

[0161] In some embodiments, the antigen-binding fragment binds to an antigen on an infectious agent, for example, a virus.

[0162] In some embodiments, the antigen-binding antibody fragment binds to an antigen on a target cell, e.g., a cancer cell or an immune cell.

[0163] In embodiments in which multiple types of fusion polypeptides with antigen-binding antibody fragments are used, the antigen-binding antibody fragments in the various types of fusion polypeptides may be capable of binding to the same epitope or may be capable of binding to distinct and non-overlapping epitopes. In some embodiments in which the epitopes are distinct and non-overlapping, the epitopes are derived from the same protein.

[0164] 4. Linker In certain embodiments, linkers are used in fusion polypeptides and / or single chain molecules such as scFc. In some embodiments, the linker is an amino acid linker. For example, a linker as used herein can contain about 1 to about 100 amino acid residues, e.g., about 1 to about 70, about 2 to about 70, about 1 to about 30, or about 2 to about 30 amino acid residues. In some embodiments, the linker contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid residues.

[0165] In certain embodiments, the linker comprises a glycine serine sequence, e.g., (G n S) m The sequence includes, for example, GGS, GGGS (SEQ ID NO:48), or GGGGS (SEQ ID NO:49).

[0166] B. Self-assembled Polypeptide Complexes In one embodiment, a self-assembled polypeptide complex is provided that includes a plurality of fusion polypeptides disclosed herein. In general, the self-assembled polypeptide complex provided includes: (a) a plurality of first fusion polypeptides, each of which includes (1) an Fc polypeptide and (2) an Fc polypeptide linked to a nanocage monomer or a subunit thereof, and the Fc polypeptide includes an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class; and (b) a plurality of second fusion polypeptides, each of which includes (1) an antigen-binding antibody fragment and (2) an antigen-binding antibody fragment linked to a nanocage monomer or a subunit thereof.

[0167] In some embodiments, the nanocage monomer is a ferritin monomer and each fusion polypeptide in the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise any ferritin heavy chain, subunit of a ferritin heavy chain, or other ferritin components capable of binding iron.

[0168] In some embodiments, the nanocage monomer or subunit thereof is a ferritin monomer subunit, and (a) each first fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, or (b) each first fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin.

[0169] In some embodiments, the self-assembling polypeptide complex comprises a total of 24 to 48 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises a total of 24 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises a total of more than 24 fusion polypeptides, for example, at least 26, at least 28, at least 30, at least 32 fusion polypeptides, at least 34 fusion polypeptides, at least 36 fusion polypeptides, at least 38 fusion polypeptides, at least 40 fusion polypeptides, at least 42 fusion polypeptides, at least 44 fusion polypeptides, at least 46 fusion polypeptides, or at least 48 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises about 32 fusion polypeptides.

[0170] In some embodiments, the self-assembled polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 first fusion polypeptides.

[0171] In some embodiments, the self-assembled polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 second fusion polypeptides.

[0172] In some embodiments, the self-assembled polypeptide complex further comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 third fusion polypeptides.

[0173] In some embodiments, the self-assembled polypeptide complex comprises about a 1:1, 1:2, 1:3, or 1:4 ratio of a first fusion polypeptide to every other fusion polypeptide.

[0174] Pharmacokinetic characteristics In certain embodiments, when administered to a subject in need of a self-assembling polypeptide complex, the provided self-assembling polypeptide complex has one or more pharmacokinetic characteristics similar to that of a reference IgG molecule (e.g., an IgG molecule that matches the class of the Fc chain in the Fc polypeptide of the first fusion polypeptide in the self-assembling polypeptide complex). In some embodiments, the pharmacokinetic characteristics discussed herein (e.g., half-life, AUC, and / or C max ) are achieved when the self-assembling polypeptide complex is administered to a human subject. In some embodiments, the range of pharmacokinetic characteristics discussed herein are achieved when the self-assembling polypeptide complex is administered via a systemic route, e.g., intravenous or subcutaneous administration.

[0175] In some embodiments, the self-assembled polypeptide complexes disclosed herein have a half-life similar to that of a reference IgG molecule. The reference IgG molecule may be, for example, an antibody from which an antigen-binding antibody fragment in a second and / or third fusion polypeptide in a self-assembled polypeptide complex is derived. For example, if an antigen-binding fragment in a second and / or third fusion polypeptide comprises a variable region from "antibody A", then in some embodiments the reference IgG molecule may be "antibody A".

[0176] In some embodiments, after administration to a subject in need of the self-assembling polypeptide complex, the self-assembling polypeptide complex remains viable for about 3 to 35 days, about 3 to about 28 days, about 3 to about 21 days, about 3 to about 14 days, about 3 to about 10 days, about 3 to about 7 days, about 3 to about 5 days, about 5 to about 35 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, about 5 to about 3 ...35 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, about 5 to about about 10 days, about 5 to about 7 days, about 7 to about 35 days, about 7 to about 28 days, about 7 to about 21 days, about 7 to about 14 days, about 7 to about 10 days, about 10 to about 35 days, about 10 to about 28 days, about 10 to about 21 days, about 10 to about 14 days, about 14 to about 35 days, about 14 to about 28 days, about 14 to about 21 days, about 21 to about 35 days, or about 21 to about 28 days. In some embodiments, after administration to a subject in need of the self-assembling polypeptide complex, the self-assembling polypeptide complex has a half-life of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, after administration to a subject in need thereof, the self-assembled polypeptide complex is detectable in serum at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days.

[0177] In some embodiments, the self-assembling polypeptide complexes disclosed herein have a bioavailability similar to that of a reference IgG molecule, e.g., an antibody from which a Fab fragment included in the self-assembling polypeptide complex is derived. For example, in some embodiments, after administration to a subject in need of the self-assembling polypeptide complex, the self-assembling polypeptide complex has a bioavailability of about 10 to about 8000 days·μg / mL, about 10 to about 7000 days·μg / mL, about 10 to about 6000 days·μg / mL, about 10 to about 5000 days·μg / mL, about 10 to about 4000 days·μg / mL, about 10 to about 3000 days·μg / mL, about 10 to about 2500 days·μg / mL, about 10 to about 1000 days·μg / mL, about 10 to about 1500 days·μg / mL, about 10 to about 100 0 days μg / mL, about 10 to about 750 days μg / mL, about 10 to about 500 days μg / mL, about 10 to about 400 days μg / mL, about 10 to about 300 days μg / mL, about 10 to about 200 days μg / mL, about 10 to about 100 days μg / mL, about 10 to about 5 0 days μg / mL, about 10 to about 25 days μg / mL, about 25 to about 8000 days μg / mL, about 25 to about 7000 days μg / mL, about 25 to about 6000 days μg / mL, about 25 to about 5000 days μg / mL, about 25 to about 4000 days μg / mL, about 2 5 to approximately 3000 days μg / mL, approximately 25 to approximately 2500 days μg / mL, approximately 25 to approximately 1000 days μg / mL, approximately 25 to approximately 1500 days μg / mL, approximately 25 to approximately 1000 days μg / mL, approximately 25 to approximately 750 days μg / mL, approximately 25 to approximately 500 days μg / mL, about 25 to about 400 days μg / mL, about 25 to about 300 days μg / mL, about 25 to about 200 days μg / mL, about 25 to about 100 days μg / mL, about 25 to about 50 days μg / mL, about 50 to about 8000 days μg / mL, about 50 to about 7000 days μg / mL, about 50 to about 6000 days, μg / mL, about 50 to about 5000 days, μg / mL, about 50 to about 4000 days, μg / mL, about 50 to about 3000 days, μg / mL, about 50 to about 2500 days, μg / mL, about 50 to about 2000 days, μg / mL, about 50 ~1500 days μg / mL, approximately 50 to approximately 1000 days μg / mL, approximately 50 to approximately 750 days μg / mL, approximately 50 to approximately 500 days μg / mL, approximately 50 to approximately 400 days μg / mL, approximately 50 to approximately 300 days μg / mL, approximately 50 to approximately 200 days μg / mL,About 50 to about 100 days·μg / mL, about 100 to about 8000 days·μg / mL, about 100 to about 7000 days·μg / mL, about 100 to about 6000 days·μg / mL, about 100 to about 5000 days·μg / mL, about 100 to about 4000 days·μg / mL, about 100 to about 3000 days·μg / mL, about 100 to about 2500 days·μg / mL, about 100 to about 1000 days·μg / mL, about 100 to about 1500 days·μg / mL, about 100 to about 1000 days·μg / mL, about 100 to about 750 days·μg / mL, about 100 to about 500 days·μg / mL, about 100 to about 400 days·μg / mL, about 100 to about 300 days·μg / mL, about 100 to about 200 days·μg / mL, about 200 to about 8000 days·μg / mL, about 200 to about 7000 days·μg / mL, about 200 to about 6000 days·μg / mL, about 200 to about 5000 days·μg / mL, about 200 to about 4000 days·μg / mL, about 200 to about 3000 days·μg / mL, about 200 to about 2000 days·μg / mL, about 200 to about 1000 days·μg / mL, about 200 to about 1500 days·μg / mL, about 200 to about 1000 days·μg / mL, about 200 to about 750 days·μg / mL, about 200 to about 500 days·μg / mL, about 200 to about 400 days·μg / mL, about 200 to about 300 days·μg / mL, about 300 to about 8000 days·μg / mL, about 300 to about 7000 days·μg / mL, about 300 to about 6000 days·μg / mL, about 300 to about 5000 days·μg / mL, about 300 to about 4000 days·μg / mL, about 300 to about 3000 days·μg / mL, about 300 to about 2500 days·μg / mL, about 300 to about 2000 days·μg / mL, about 300 to about 1500 days·μg / mL, about 300 to about 1000 days·μg / mL, about 300 to about 750 days·μg / mL, about 300 to about 500 days·μg / mL, about 300 to about 400 days·μg / mL, about 400 to about 8000 days·μg / mL, about 400 to about 7000 days·μg / mL, about 400 to about 6000 days·μg / mL, about 400 to about 5000 days·μg / mL, about 400 to about 4000 days·μg / mL, about 400 to about 3000 days·μg / mL, about 400 to about 2500 days·μg / mL, about 400 to about 2000 days·μg / mL, about 400 to about 1500 days·μg / mL, about 400 to about 1000 days·μg / mL, about 400 to about 750 days·μg / mL, about 400 to about 500 days·μg / mL, about 500 to about 8000 days·μg / mL,About 500 to about 7000 days·μg / mL, about 500 to about 6000 days·μg / mL, about 500 to about 5000 days·μg / mL, about 500 to about 4000 days·μg / mL, about 500 to about 3000 days·μg / mL, about 500 to about 2500 days·μg / mL, about 500 to about 2000 days·μg / mL, about 500 to about 1500 days·μg / mL, about 500 to about 1000 days·μg / mL, about 500 to about 750 days·μg / mL, about 750 to about 8000 days·μg / mL, about 750 to about 7000 days·μg / mL, about 750 to about 6000 days·μg / mL, about 750 to about 5000 days·μg / mL g / mL, about 750 to about 4000 days·μg / mL, about 750 to about 3000 days·μg / mL, about 750 to about 2500 days·μg / mL, about 750 to about 2000 days·μg / mL, about 750 to about 1500 days·μg / mL, about 750 to about 1000 days·μg / mL, about 1000 to about 8000 days·μg / mL, about 1000 to about 7000 days·μg / mL, about 1000 to about 6000 days·μg / mL, about 1000 to about 5000 days·μg / mL, about 1000 to about 4000 days·μg / mL, about 1000 to about 3000 days·μg / mL, about 1000 to about 2500 days·μg / mL, about 1000 to about 2000 days·μg / mL, about 1000 to about 1500 days·μg / mL, about 1500 to about 8000 days·μg / mL, about 1500 to about 7000 days·μg / mL, about 1500 to about 6000 days·μg / mL, about 1500 to about 5000 days·μg / mL, about 1500 to about 4000 days·μg / mL, about 1500 to about 3000 days·μg / mL, about 1500 to about 2500 days·μg / mL, about 1500 to about 2000 days·μg / mL, about 2000 to about 8000 days·μg / mL, about 2000 to about 7000 days·μg / mL, about 2000 to about 6000 days·μg / mL, About 2000 to about 5000 days·μg / mL, about 2000 to about 4000 days·μg / mL, about 2000 to about 3000 days·μg / mL, about 2000 to about 2500 days·μg / mL, about 2500 to about 8000 days·μg / mL, about 2500 to about 7000 days·μg / mL, about 2500 to about 6000 days·μg / mL, about 2500 to about 5000 days·μg / mL, about 2500 to about 4000 days·μg / mL, about 2500 to about 3000 days·μg / mL, about 3000 to about 8000 days·μg / mL, about 3000 to about 7000 days·μg / mL, about 3000 to about 6000 days·μg / mL,About 3000 to about 5000 days μg / mL, about 3000 to about 4000 days μg / mL, about 4000 to about 8000 days μg / mL, about 4000 to about 7000 days μg / mL, about 4000 to about 6000 days μg / mL, about 4000 to about 500 0 days μg / mL, about 5000 to about 8000 days μg / mL, about 5000 to about 7000 days μg / mL, about 5000 to about 6000 days μg / mL, about 6000 to about 8000 days μg / mL, or It has an area under the curve (AUC) of about 7000 to about 8000 days μg / mL. In some embodiments, after administration to a subject in need thereof, the self-assembling polypeptide complex has an AUC of at least 10 days·μg / mL, at least 25 days·μg / mL, at least 50 days·μg / mL, at least 100 days·μg / mL, at least 200 days·μg / mL, at least 300 days·μg / mL, at least 400 days·μg / mL, at least 500 days·μg / mL, at least 750 days·μg / mL, at least 1000 days·μg / mL, at least 1500 days·μg / mL, at least 2000 days·μg / mL, at least 2500 days·μg / mL, at least 3000 days·μg / mL, at least 4000 days·μg / mL, at least 5000 days·μg / mL, at least 6000 days·μg / mL, at least 7000 days·μg / mL, or at least 8000 days·μg / mL.

[0178] In some embodiments, the self-assembling polypeptide complexes disclosed herein have a bioavailability similar to that of a reference IgG molecule. For example, in some embodiments, after administration to a subject in need thereof, the self-assembling polypeptide complex has a bioavailability of about 10 μg / mL to about 750 mg / mL, about 25 μg / mL to about 750 mg / mL, about 50 μg / mL to about 750 mg / mL, about 75 μg / mL to about 750 mg / mL, about 100 μg / mL to about 750 mg / mL, about 250 μg / mL to about 750 mg / mL, about 500 μg / mL to about 750 mg / mL, about 750 μg / mL to about 750 mg / mL, about 1 mg / mL to about 750 mg / mL, or about 2 mg / mL to about 750 mg / mL. mL, about 10 mg / mL to about 750 mg / mL, about 25 mg / mL to about 750 mg / mL, about 50 mg / mL to about 750 mg / mL, about 75 mg / mL to about 750 mg / mL, about 100 mg / mL to about 750 mg / mL, about 250 mg / mL to about 750 mg / mL mL, about 500mg / mL to about 750mg / mL, about 10μg / mL to about 500mg / mL, about 25μg / mL to about 500mg / mL, about 50μg / mL to about 500mg / mL, about 75μg / mL to about 500mg / mL, about 100μg / mL to about 500mg / m L, about 250μg / mL to about 500mg / mL, about 500μg / mL to about 500mg / mL, about 750μg / mL to about 500mg / mL, about 1mg / mL to about 500mg / mL, about 10mg / mL to about 500mg / mL, about 25mg / mL to about 500mg / m L, about 50 mg / mL to about 500 mg / mL, about 75 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 10 μg / mL to about 250 mg / mL, about 25 μg / mL to about 250 mg / mL , about 50μg / mL to about 250mg / mL, about 75μg / mL to about 250mg / mL, about 100μg / mL to about 250mg / mL, about 250μg / mL to about 250mg / mL, about 500μg / mL to about 250mg / mL, about 750μg / mL to about 250mg / mL mL, about 1 mg / mL to about 250 mg / mL, about 10 mg / mL to about 250 mg / mL, about 25 mg / mL to about 250 mg / mL, about 50 mg / mL to about 250 mg / mL, about 75 mg / mL to about 250 mg / mL, about 100 mg / mL to about 250 mg / mL,About 10 μg / mL to about 100 mg / mL, about 25 μg / mL to about 100 mg / mL, about 50 μg / mL to about 100 mg / mL, about 75 μg / mL to about 100 mg / mL, about 100 μg / mL to about 100 mg / mL, about 250 μg / mL to about 100 mg / mL, about 500 μg / mL to about 100 mg / mL, about 750 μg / mL to about 100 mg / mL, about 1 mg / mL to about 100 mg / mL, about 10 mg / mL to about 100 mg / mL, about 25 mg / mL to about 100 mg / mL, about 50 mg / mL to about 100 mg / mL, about 75 mg / mL to about 100 mg / mL, About 10μg / mL to about 75mg / mL, about 25μg / mL to about 75mg / mL, about 50μg / mL to about 75mg / mL, about 75μg / mL to about 75mg / mL, about 100μg / mL to about 75mg / mL, about 250μg / mL to about 75mg / mL, about 500μg / mL to about 75mg / mL, about 750μg / mL to about 75mg / mL, about 1mg / mL to about 75mg / mL, about 10mg / mL to about 75mg / mL, about 25mg / mL to about 75mg / mL, about 50mg / mL to about 75mg / mL, about 10μg / mL to about 50mg / mL, about 25μg / mL to about 50mg / mL g / mL, about 50μg / mL to about 50mg / mL, about 75μg / mL to about 50mg / mL, about 100μg / mL to about 50mg / mL, about 250μg / mL to about 50mg / mL, about 500μg / mL to about 50mg / mL, about 750μg / mL to about 50mg / mL, about 1mg / mL to about 50mg / mL, about 10mg / mL to about 50mg / mL, about 25mg / mL to about 50mg / mL, about 10μg / mL to about 25mg / mL, about 25μg / mL to about 25mg / mL, about 50μg / mL to about 25mg / mL, about 75μg / mL to about 25mg / mL, about 100μg / mL L to about 25 mg / mL, about 250μg / mL to about 25 mg / mL, about 500μg / mL to about 25 mg / mL, about 750μg / mL to about 25 mg / mL, about 1mg / mL to about 25 mg / mL, about 10mg / mL to about 25 mg / mL, about 10μg / mL to about 10mg / mL, about 25μg / mL to about 10mg / mL, about 50μg / mL to about 10mg / mL, about 75μg / mL to about 10mg / mL, about 100μg / mL to about 10mg / mL, about 250μg / mL to about 10mg / mL, about 500μg / mL to about 10mg / mL, about 750μg / mL to about 10mg / mL,Approximately 1 mg / mL to approximately 10 mg / mL, approximately 10 μg / mL to approximately 1 mg / mL, approximately 25 μg / mL to approximately 1 mg / mL, approximately 50 μg / mL to approximately 1 mg / mL, approximately 75 μg / mL to approximately 1 mg / mL , about 100 μg / mL to about 1 mg / mL, about 250 μg / mL to about 1 mg / mL, about 500 μg / mL to about 1 mg / mL, about 750 μg / mL to about 1 mg / mL, about 10 μg / mL to about 7 50μg / mL, approximately 25μg / mL to approximately 750μg / mL, approximately 50μg / mL to approximately 750μg / mL, approximately 75μg / mL to approximately 750μg / mL, approximately 100μg / mL to approximately 750μg / mL , about 250μg / mL to about 750μg / mL, about 500μg / mL to about 750μg / mL, about 10μg / mL to about 500μg / mL, about 25μg / mL to about 500μg / mL, about 50μg / mL ~ approx. 500μg / mL, approx. 75μg / mL ~ approx. 500μg / mL, approx. 100μg / mL ~ approx. 500μg / mL, approx. 250μg / mL ~ approx. 500μg / mL, approx. 10μg / mL ~ approx. 2 50μg / mL, approximately 25μg / mL to approximately 250μg / mL, approximately 50μg / mL to approximately 250μg / mL, approximately 75μg / mL to approximately 250μg / mL, approximately 100μg / mL to approximately 250μg / mL , about 10μg / mL to about 100μg / mL, about 25μg / mL to about 100μg / mL, about 50μg / mL to about 100μg / mL, about 75μg / mL to about 100μg / mL, about 10μg / m L ~ about 75 μg / mL, about 25 μg / mL to about 75 μg / mL, about 50 μg / mL to about 75 μg / mL, about 10 μg / mL to about 50 μg / mL, about 25 μg / mL to about 50 μg / mL, or Maximum concentration of approximately 10 μg / mL to approximately 25 μg / mL (C, max In some embodiments, after administration to a subject in need thereof, the self-assembling polypeptide complex has a maximum concentration (C) of at least 10 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 250 μg / mL, at least 500 μg / mL, at least 750 μg / mL, at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL, at least 100 mg / mL, at least 250 mg / mL, at least 500 mg / mL, or at least 750 mg / mL.max ).

[0179] Functional Effects In certain embodiments, the self-assembling polypeptide complexes provided are capable of antibody-dependent cellular phagocytosis (ADCP). In some embodiments, ADCP is induced at a level of internalization of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the target. Methods for measuring ADCP are known in the art and include, for example, in vitro assays utilizing macrophage cell lines and targets.

[0180] C. Treatment method In one aspect, methods are provided that may be useful for treating, alleviating, or preventing a disease or condition (e.g., an infectious disease, cancer, or an autoimmune disease) generally comprising administering to a subject a composition comprising a self-assembling polypeptide complex of the present disclosure.

[0181] In some embodiments, the subject is a mammal, such as a human.

[0182] Compositions for administration to a subject generally comprise the self-assembling polypeptide complexes disclosed herein. In some embodiments, such compositions further comprise a pharma- ceutically acceptable excipient.

[0183] The compositions can be formulated for administration by any of a variety of routes, including systemic routes (eg, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration). EXAMPLES

[0184] Example 1. Expression and analysis of representative multibody In this example, the (Gly n -Ser) mMultabodies (MBs) were formed by fusion proteins generated by fusing the human ferritin light chain (hFTL, SEQ ID NO:1), the N-terminal fragment (residues 1-90) of hFTL (N_hFTL, SEQ ID NO:2), or the C-terminal fragment (residues 91-175) of hFTL (C_hFTL, SEQ ID NO:3) with a single-chain Fab (scFab) and / or a fragment crystallizable region (Fc) or a single-chain Fc dimer (scFc) via a linker, such as a peptide linker. The different formats generated are shown in Figure 1.

[0185] T-01 MB: Genes encoding fusion proteins (1) scFab of HIV neutralizing antibody PGDM1400 fused to the N-terminus of hFTL (PGDM1400-hFTL, SEQ ID NO: 27), (2) scFc fused to the N-terminus of N_hFTL (scFc-N_hFTL, SEQ ID NO: 34), (3) scFab of HIV neutralizing antibody N49P7 fused to the N-terminus of C_hFTL (N49P7-C_hFTL, SEQ ID NO: 28), and (4) scFab of HIV neutralizing antibody 10E8v4 fused to the N-terminus of C_hFTL (10E8v4-C_hFTL, SEQ ID NO: 30) were prepared, mixed in a molar ratio of 4:2:1:1, and transiently transfected into HEK293F cells to produce and form T-01 MB. See FIG. 1B.

[0186] T-02 MB: Genes encoding fusion proteins (1) PGDM1400-hFTL (SEQ ID NO: 27), (2) scFc-N_hFTL (SEQ ID NO: 34), (3) scFab of anti-CD4 antibody ibalizumab (iMab) fused to the N-terminus of C_hFTL (iMab-C_hFTL, SEQ ID NO: 31), and (4) 10E8v4-C_hFTL (SEQ ID NO: 30) were prepared, mixed in a molar ratio of 4:2:1:1, and transiently transfected into HEK293F cells for the production and formation of T-02 MB. See FIG. 1B.

[0187] T-01 MB.v2: Genes encoding fusion proteins (1) PGDM1400-hFTL (SEQ ID NO: 27), (2) scFab of N49P7 fused to the N-terminus of N_hFTL (N49P7-N_hFTL (SEQ ID NO: 29), and (3) Fc monomer fused to the C-terminus of 10E8-C_hFTL (10E8v4-C_hFTL-Fc, SEQ ID NO: 32) were prepared and mixed in a molar ratio of 3:1:1 and transiently transfected into HEK293F cells for the production and formation of T-01 MB.v2. See FIG. 1D. (See "T-01" above). As described for "MB", 10E8-C_hFTL contains the scFab of the HIV neutralizing antibody 10E8v4 fused to the N-terminus of C_hFTL. Thus, the construct 10E8v4-C_hFTL-Fc (SEQ ID NO: 32) contains a C-half ferritin with a Fab3 at the N-terminus of the C-half ferritin, and an Fc monomer at the C-terminus of the C-half ferritin.

[0188] The multabodies described in this example had wild-type (WT) Fc or engineered IgG1 Fc. Such engineered IgG1 Fc contained any one or more of the following mutations (according to the EU numbering scheme): L234A, L235A, K288A, I253V, I253A, P329G, M428L, N434S, or any combination thereof. For example, T-01 MB IgG1 K288A had an engineered IgG1 Fc with a K288A mutation, T-01 MB IgG1 LALAP had an engineered IgG1 Fc with L234A, L235A, and P329G mutations, and T-01 MB.v2 IgG1 LS had an engineered IgG1 Fc with M428L and N434S mutations.

[0189] Multabodies were purified using Protein A affinity chromatography, optionally followed by Protein L affinity chromatography. Fractions containing Multabodies were concentrated and further purified by size exclusion chromatography (SEC) in sodium phosphate buffer. After SEC purification, negative staining electron microscopy (EM) and / or SEC with multi-angle light scattering (SEC-MALS) were used to assess the size of the Multabodies formed.

[0190] Example 2. Binding of multibodies to Fc receptors determined by biolayer interferometry The binding kinetics and affinity of multibodies containing different Fc mutations to Fc receptors—human Fcγ receptor I (hFcγRI), hFcγRIIa, and hFcγRIIb—were determined by biolayer interferometry (BLI) using an Octet RED96 BLI system (Pall ForteBio).

[0191] Briefly, His-tagged Fc receptors were loaded onto Ni-NTA biosensors to reach a signal response of 0.8 nm. Association rates were measured by transferring the loaded biosensors to wells containing serial dilutions of test Multabodies (20-10-5-2.5-1.25-0.65 nM) or IgG1 controls (250-125-62.5-31.2-15.6-7.8 nM) with a contact time of 180 seconds. The IgG1 control is a cocktail of PGDM1400, N49P7, and 10E8v4 antibodies, all with wild-type IgG1 backbone. To assess the potential of Multabodies to undergo endosomal recycling, their binding to the hFcRn / β2-microglobulin complex was measured at both physiological pH (7.4) and acidic pH (5.6).

[0192] The IgG1 backbone Fc mutations evaluated in the multibody include K288A, I253V, and I253A, which reduce antibody binding to FcRn, P329G, LALA (L234A, L235A), and LALAP (L234A, L235A, and P329G), which reduce antibody binding to FcγRs, and combinations thereof. (Numbering follows the EU numbering scheme.)

[0193] Representative examples of relevant segments of the resulting sensorgrams are provided in Figures 3A, 3B, 3C, 3D, 3E, and 3F. on , k off , and the equilibrium dissociation constant of the obtained multibody (K D ) are summarized in Tables 1 and 2.

[0194] At acidic pH (5.6), T-01 MB with wild-type IgG1 Fc binds to FcRn with over 1000-fold affinity compared to the IgG1 control; the same is observed for T-01 MB with K288A, I253V, P329G, LALA, LALAP, K288A+P329G, or K288A+LALAP IgG1 Fc mutations. T-01 MB with I253A or I253A+LALAP IgG1 Fc mutations have similar binding ability to FcRn at pH 5.6 compared to the IgG1 control. At physiological pH (7.4), T-01 MB with wild-type IgG1 Fc, P329G IgG1 Fc mutation, LALA IgG1 Fc mutation, or LALAP IgG1 Fc mutation show measurable binding to FcRn.

[0195] T-01 MB with wild type IgG1 Fc binds to hFcγRI, hFcγRIIa, and hFcγRIIb with over 1000-fold higher affinity compared to the IgG1 control. Multabodies with P329G, LALA, or LALAP IgG1 Fc mutation(s) show reduced or no binding to the Fcγ receptors tested.

[0196] T-01 MB.v2 with wild-type IgG1 Fc or LS Fc mutations has an FcRn binding profile more similar to IgG1, with comparable binding ability to human FcRn at acidic pH and no binding at physiological pH. Furthermore, T-01 MB.v2 with IgG1 Fc or LS mutations, like T-01 MB containing the LALAP mutation, shows reduced binding to the high affinity FcγRI and no binding to the low affinity Fcγ receptors tested.

[0197] Among the Fc mutations and mutation combinations tested, the I253A+LALAP IgG1 Fc mutation combination modulates the Fc receptor binding profile of the multibody (T-01 MB format) to be IgG1-like. [Table 1] [Table 2]

[0198] Example 3. Target Binding of Multabody Determined by Biolayer Interferometry The binding kinetics and affinity of PGDM1400, N49P7, 10E8v4, or iMab contained as Fab in the multibody to their respective targets were determined by BLI using an Octet RED96 BLI system (Pall ForteBio).

[0199] The experiment was performed similarly as described in Example 2, except that the Hi-tagged targets for PGDM1400, N49P7, 10E8v4, and iMab, respectively - BG5050 SOSIP.664 D368R, gp120 subunit 93TH057, gp41 membrane proximal external region (MPER), and soluble CD4- were loaded onto Ni-NTA biosensors to reach a signal response of 0.8 nm. The loaded biosensors were then titrated with various concentrations of test Multabody, PGDM1400, N49P7, 10E8v4, or iMab antibodies (with wild-type IgG1 backbone), or a cocktail (IgG1 control) of PGDM1400, N49P7, and 10E8v4 antibodies (all with wild-type IgG1 backbone).

[0200] Representative examples of relevant segments of the resulting sensorgrams are provided in Figures 4A, 4B, 4C, 4D, and 4E. on , k off , and the equilibrium dissociation constant of the obtained multibody (K D The determined values ​​for are summarized in Tables 3 and 4. [Table 3] [Table 4]

[0201] Example 4. Pharmacokinetics of Multabody in Mice Pharmacokinetic analysis of multibodies with wild-type or engineered IgG1 Fc was performed in mice.

[0202] Test multibody or IgG1 control was administered at a dose of 5 mg / kg to five CB17 / Icr-Prkdc mice on day 0. scid / IcrIcoCrl immunodeficient (SCID) mice / group were injected with 10 mg / kg / IcrIcoCrl. The IgG1 control was a cocktail of PGDM1400, N49P7, and 10E8v4 antibodies, all with a wild-type IgG1 backbone. Serum samples were collected every 2 days for 9 days, starting on day 1. On day 10, a booster dose of 5 mg / kg was administered and serum samples were collected on days 11 and 15.

[0203] Additionally, multabodies containing wild-type Fc, LALAP+I253A IgG1 Fc mutation combination, or M428L+N434S (LS) Fc mutation combination were subcutaneously injected into NOD / Shi-scid / IL-2Rγnull immunodeficient (NCG) mice (3 mice / group) at a single dose of 5 mg / kg. Blood samples were taken at multiple time points after injection. An IgG1 control was tested in parallel.

[0204] Circulating Multabody levels were assessed by ELISA. Briefly, 96-well plates were coated with 50 mL of His-tagged antigen recognized by the Fab in Multabody at 0.5 mg / mL. Serum / blood samples were diluted and added to the wells. Bound drug was detected using HRP-Protein A as the secondary molecule. Chemiluminescent signals were quantified using a microplate reader. A calibration curve with standard protein dilutions was prepared.

[0205] Figures 5B-5E and 6B show plots of plasma concentration over time for the tested Multabodies. The LALAP+I235A, LALAP+K288A, and K288A+P329G mutation combinations were able to restore serum levels of T-01 MB to similar to the IgG1 control; the LS mutation combination was able to restore serum levels of T-01 MB.v2 to similar to the IgG1 control. Thus, T-01 MB IgG1 LALAP I235A, T-01 MB IgG1 LALAP K288A, T-01 MB IgG1 K288A P329G, and T-01 MB.v2 IgG1 LS exhibit antibody-like pharmacokinetics or favorable pharmacokinetic profiles.

[0206] Example 5. Evaluation of multibody-induced antibody-dependent cellular phagocytosis The potential of selected multibodies to induce antibody-dependent cellular phagocytosis (ADCP) was assessed using the THP-1 cell line.

[0207] Red fluorescent FluoSpheres NeutrAvidin microspheres were coated with biotinylated 93TH057 gp120 (the antigen of N49P7) and incubated with T-01 MB IgG1 LALAP I253A or T-01 MB.v2 IgG1 LS, or an IgG1 cocktail of PGDM1400, N49P7, and 10E8v4 IgG1 antibodies at various concentrations for 2 h at 37 °C, followed by incubation with 5 × 10 4 200 μL of THP-1 cells were added at cells / well. After 16 hours, cells were pelleted and washed with PBS. Live / Dead Fixable Violet Stain was used to determine cell viability. PBS-washed cells were fixed with 2% paraformaldehyde for 20 minutes at room temperature, pelleted, and washed once with FACS buffer (PBS+10% FBS, 0.5 mM EDTA). Cells were then analyzed using a BD LSR II flow cytometer and data analyzed using FlowJo. IgG1 and Multabody controls with no affinity for 93TH057 gp120 were tested in parallel. Fc-mediated internalization was blocked using an FcR binding inhibitor antibody (Invitrogen, 14-9161-73).

[0208] The results are shown in Figure 7. Phagocytosis was quantified and expressed as the percentage increase in internalization of 93TH057-coated microspheres compared to uncoated microspheres. T-01 MB.v2 IgG1 LALAP I253A induces dose-dependent ADCP comparable to the IgG1 control despite low binding to FcγRI (see Example 2).

[0209] Example 6. Evaluation of multibody-mediated neutralization of HIV-1 The ability of selected multibodies to neutralize HIV-1 was assessed using the TZM-bl assay, which measures HIV-1 neutralization as a function of the reduction in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped virus.

[0210] Briefly, HIV-1 pseudotyped viruses were generated using the HIV-1 subtype B backbone NL4-3.Luc.R, a plasmid encoding a full-length Env clone. - E plasmid was generated by co-transfecting 293T cells with the test Multabody. The test Multabody, the Fab antibodies included in the Multabody, IgG1 control-1 (a cocktail of PGDM1400, N49P7, and 10E8v4 IgG1 antibodies), IgG1 control-2 (a cocktail of PGDM1400, iMab, and 10E8v4 IgG1 antibodies), or the N6 / PGDM1400x10E8v4 trispecific antibody (directed against the CD4bs, V1V2 apex, and MPER binding sites) were incubated with 10–15% tissue culture infectious dose of pseudovirus for 1 h at 37°C before incubation with pseudovirus-transfected cells for 44–72 h. Virus neutralization was monitored by adding Britelite+ reagent (PerkinElmer) to the cells and measuring luminescence in relative light units (RLU) using a Synergy Neo2 multimode assay microplate reader. Test samples were assayed against a single pseudovirus or a panel of 14 or 25 pseudoviruses (14 or 25 PsV panels). The 25-PsV panel includes strains in the 14-PsV panel and adds 11 HIV-1 strains that are highly resistant to PDGM1400 to the 14-PsV panel. Thus, the 25-PsV panel is a panel that contains the PsV variants that are resistant to PDGM1400 IgG neutralization (cutoff IC 50 contains 56% of the total IgG (set at 10 μg / mL).

[0211] Exemplary results are shown in Figures 8A, 8B, 8C, and 8D, and IC 50The determined values ​​for and breadth of neutralization are summarized in Tables 5 and 6. The multibodies were determined using the IC50 and IC60 values ​​for the IgG cocktail and trispecific antibodies, respectively. 50 Compared with the values, the median IC 50 The neutralization profile of antibodies N49P7 and 10E8v4 is superior to other multibodies, and T-01 MB.v2 achieved 100% neutralization in the 25-PsV panel.

[0212] When tested against an expanded multi-clade panel of 118 PsV, T-01 MB.v2 surpassed the pan-neutralization breadth (100% viral coverage, cut-off IC ) of the corresponding IgG cocktail. 50 The IC50 of 0.001 μg / mL was set at 10 μg / mL), but showed significant neutralization potency (Figures 21C-D, 22A, and Table 9). Specifically, the IgG cocktail and T-01 MB each had an IC50 of 0.001 μg / mL. 50 The IC50 values ​​for T-01 MB.v2 were still only 0.001 μg / mL, whereas the IC50 values ​​for T-01 MB.v2 were only 0.001 μg / mL. 50 Notably, the multibody neutralized 50% of PsV with a median IC value of only 0.0009 μg / mL (0.4 pM) (Figure 21C). 50 values, thus achieving pan-neutralization 32-fold and 490-fold more potent in mass and molar concentrations, respectively, compared to the IgG cocktail ( FIG. 21D ). In addition, the IC 80 confirmed a tendency for neutralization superior to both individual IgG and IgG cocktails, with a median IC of 0.005 μg / mL (2.2 pM). 80 The IgG neutralization levels were 96% for all virus strains tested (Figures 21C-D, Figure 22A, and Table 9). Importantly, the multabody also blocked infection of primary peripheral blood mononuclear cells (PBMCs) with a replication-competent CXCR4-tropic HIV-1 IIIB strain (Figure 22B), with greater potency than a matched IgG mixture, and without any effect on cell viability (Figure 22C). [Table 5] [Table 6]

[0213] Example 7. Evaluation of inhibition of HIV-1 infection by multibodies The ability of selected multibodies to inhibit HIV-1 infection was assessed using human peripheral blood mononuclear cells (PBMCs).

[0214] Briefly, PBMCs were obtained from three healthy blood donors and activated with phytohemagglutinin (PHA) in the presence of recombinant human IL-2 in complete RPMI medium supplemented with 10% fetal bovine serum (FBS) for 72 h prior to HIV-1 infection. Laboratory CXCR4-tropic HIV-1 isolate IIIB was added in triplicate to the activated PBMCs after 1 h of incubation at room temperature with the test Multabody or IgG1 control. The IgG1 control is a cocktail of PGDM1400, N49P7, and 10E8v4 antibodies, all with a wild-type IgG1 backbone. Infected cells were cultured in the presence or absence of the test Multabody or antibody control at doses ranging from 0.01 to 10 μg / mL. The level of HIV-1 replication was assessed on day 7 postinfection by measuring the extracellular release of p24 Gag protein in cell-free culture supernatants using a highly sensitive AlphaLISA p24 detection kit on a BioTEK Synergy Plate Reader according to the manufacturer's protocol. Cell viability was also assessed on day 7 postinfection by fixing cells in 2% PFA and absolute cell numbers were counted by flow cytometry using a BD LSRFortessa (Becton Dickinson).

[0215] Exemplary results are shown in Figures 9A and 9B. Multabodies (T-01 MB and T-01 MB.v2) were able to inhibit infection of primary PBMCs by the replication-competent CXCR4-tropic HIV-1 isolate IIIB, with enhanced potency compared to the IgG1 control, and without any effect on cell viability.

[0216] Example 8. Characterization of Thermal Stability of Multabodies Melting temperatures (T m ) and agglomeration temperature (T agg ) was determined using a UNit system. Samples were concentrated to 1.0 mg / mL and subjected to a thermal gradient from 25° C. to 95° C. in 1° C. increments. T m is obtained by measuring the barycentric mean fluorescence, T agg was determined as the temperature at which a 50% increase in static light scattering at a wavelength of 266 nm was observed compared to the baseline. The mean and standard error of three independent measurements were calculated using UNit analysis software. Table 7 shows the T m and T agg To summarize:

[0217] The stability of Multabodies was further analyzed under accelerated conditions. Samples were concentrated to 10 mg / mL and incubated at 40° C. for 4 weeks. Each week, the percentage of properly folded protein was calculated based on the soluble content from SEC. Multabodies were highly stable under these conditions, with more than 70% of the samples remaining soluble for 30 days. (See FIG. 10A).

[0218] Additionally, samples from before (week 0) and after (week 4) the incubation period were evaluated in a PsV neutralization assay to compare the biological function of the molecules. Stability was further confirmed by the slight loss of neutralization potency at week 4 compared to potency at week 0. (See FIG. 10B).

[0219] The tested Multabodies have similar thermal stability compared to the reference molecules and are stable for at least 4 weeks when stored at 40° C. with minimal loss of neutralizing potency. [Table 7] Example 9. Engineering pan-HIV-1 neutralization potency via multispecific antibody avidity summary Deep mining of the B-cell repertoire of HIV-1-infected individuals has led to the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). However, it remains unclear whether any such bNAb alone is broad and potent enough to be deployed therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule exhibited a remarkable median IC of 0.0009 μg / mL with a cutoff of 4 μg / mL. 50 The antibody demonstrated high antibody titers and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118-isolates). This is a 32-fold enhancement in virus neutralization potency compared to the corresponding cocktail of HIV-1 bNAbs. Importantly, Fc incorporation into the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is relevant for indications where multiple specificities and avidities are simultaneously utilized to mediate optimal bioactivity. The high genetic diversity of HIV-1 remains a major barrier to the development of therapeutic agents for prevention and treatment. Here, we describe the design of an antibody platform that allows the assembly of highly avid, multispecific molecules that simultaneously target the most conserved epitopes on the HIV-1 envelope glycoprotein. The multivalent and multispecific combination translated into exceptional neutralization potency and pan-neutralization of HIV-1 strains, surpassing that of the most potent anti-HIV broadly neutralizing antibody cocktails. Introduction Despite decades of research, no effective vaccines or treatments exist for human immunodeficiency virus type I (HIV-1). However, the fact that a small proportion of HIV-1-infected individuals develop antibodies with exceptional neutralizing potency across circulating HIV-1 isolates highlights the possibility of antibody-mediated control of HIV-1. Since the isolation of the first generation of broadly neutralizing antibodies (bNAbs) 2F5 (1), 4E10 (2, 3), 2G12 (4), and b12 (5, 6), the number of bNAbs has increased dramatically with the implementation of new techniques such as Env-specific single B cell sorting (7-9), antibody cloning and high-throughput neutralization assays (10-13), and more recently proteomic deconvolution (14). Currently, several HIV-1 bNAbs have been described that primarily target six conserved sites on the trimeric HIV envelope glycoprotein (Env), including the V1 / V2 loop at the trimer apex, the V3 loop glycan, the CD4 binding site (CD4bs), the gp120-g41 interface, the Env silent face, and the membrane-proximal external region (MPER) ( 7 , 9 , 11 – 20 ). Interest in bNAbs as therapeutics in the fight against HIV-1 stems from the potent antiviral activity observed in challenge studies in macaques (21–25) and humanized mice (26–29), as well as the reduction in viremia achieved in infected humans after infusion of bNAbs (30–34). Furthermore, antibodies have important advantages compared to oral antiretroviral therapy (ART), in that they have a longer circulating half-life and can form immune complexes that enhance host immunity against the virus. These observations have led to the clinical evaluation of antibody-based therapies to confer protection against HIV-1 acquisition by passive administration of bNAbs (35), as well as efforts to control and / or eliminate HIV-1 in infected individuals (31–33). Recent antibody-mediated prophylaxis (AMP) trials have explored the ability of bNAb VRC01 to confer passive immunity against HIV-1. These studies proposed antibody breadth and potency, estimated from TZM-bl neutralization assays, as valid predictors of antibody efficacy in humans. Specifically, an IC of less than 1 μg / ml was proposed as the efficacy threshold that a biotherapeutic needs to achieve to confer protection against a specific HIV-1 strain. 80 The authors established a threshold for broad coverage of IgG antibodies against 100% of HIV-1 strains in the study (35). VRC01 only met that threshold against 30% of HIV-1 strains in testing and therefore failed to confer broad-spectrum protection, highlighting the critical need for more potent, broad-acting molecules. Such broad coverage can be achieved by administration of multiple bNAbs, but despite recent IgG engineering efforts (36-40), potency may still limit the therapeutic effectiveness of antibody cocktails. Here, we overcome the enormous sequence diversity of HIV-1 with extraordinary neutralizing potency by engineering human apoferritin subunits to promote multimerization of three distinct HIV-1 bNAbs on a single molecule. The resulting multispecific, multi-affinity antiBODYs (Multabodies) have a median IC of 0.0009 μg / mL (0.4 pM). 50 Pan-neutralization (100% viral coverage) could be achieved at 100% IgG values. The multibody design described herein represents a robust and potent plug-and-play platform for multimerizing antibodies to enhance their neutralization of HIV-1 across the broadest range of isolates. Materials and Methods Expression and purification of Fab-only apoferritin multimers. Genes encoding the light chain of human apoferritin and scFab-human apoferritin fusions were synthesized and cloned into the pHLsec expression vector by GeneArt (Life Technologies). 200 mL of HEK293F cells (Thermo Fisher Scientific) were incubated with 0.8 × 10 6Cells were seeded in Freestyle expression medium at a density of 1000 cells / mL and incubated at 37 °C, 8% CO2, and 70% humidity in a Multitron Pro shaker (Infors HT) with 125 rpm shaking. Within 24 h of seeding, cells were transiently transfected with 50 μg of filtered DNA preincubated for 10 min at room temperature (RT) with the transfection reagent FectoPRO (Polyplus Transfections) in a 1:1 ratio. scFab-human apoferritin and plasmids encoding human apoferritin were mixed in ratios of 1:4, 1:1, 4:1, and 1:0. After 6-7 days, cell suspensions were harvested by centrifugation at 5000 × g for 15 min, and the supernatant was filtered through a 0.22 μm Steritop filter (EMD Millipore). Particles were purified to Fab by affinity chromatography and eluted after washing. Fractions containing protein were pooled, concentrated and loaded onto a Superose 6 10 / 300GL size exclusion column (GE Heathcare) in 20 mM sodium phosphate, pH 8.0, 150 mM NaCl. Multabody design, expression, and purification. Genes encoding scFab and scFc fragments linked to half-ferritin were generated by deletion of residues 1-90 (C-ferritin) and 91-175 (N-ferritin) of the light chain of human apoferritin. Furthermore, protein L binding specificity for iMab-C-ferritin was abolished by site-directed mutagenesis of alanine 12 of the antibody light chain to a proline residue (69). Transient transfection of T-01 MB in HEK 293F cells was obtained by mixing 66 μg of plasmids PGDM1400 scFab-human apoferritin:scFc-N-Ferritin:N49P7 scFab-C-ferritin:10E8v4 scFab-C-ferritin in a ratio of 4:2:1:1. For T-02 MB, plasmid N49P7 scFab-C-ferritin was replaced with iMab scFab-C-ferritin. For T-01 MB.v2, 63 μg of plasmid PGDM1400 scFab-human apoferritin:N49P7 scFab-N-ferritin:10E8v4 scFab-C-ferritin-Fc (3:1:1 ratio) was used. The DNA mixture was filtered and incubated at room temperature with 60 μl FectoPRO before adding to the cell culture. Based on the hetero-oligomerization required to drive self-assembly, purification of the four-component Multabody was achieved by two-step affinity purification: Protein A HP column (GE Healthcare) with 20 mM Tris pH 8.0, 3 M MgCl2 and 10% glycerol elution buffer (Fc binding) and Protein L (GE Healthcare) (PGDM1400 binding, since 10E8 and N49P7 did not bind Protein L and the A12P mutation disrupted the iMab-Protein L binding). A buffer exchange step was performed between both affinity chromatography steps using a PD-10 desalting column (GE Healthcare). The protein-containing fractions were concentrated and further purified by gel filtration on a Superose 6 10 / 300GL column (GE Healthcare) in 20 mM sodium phosphate pH 8.0, 150 mM NaCl. Negative staining electron microscopy. Three microliters of Multabody at a concentration of approximately 0.02 mg / mL was added to carbon-coated copper grids for 30 seconds and stained with 3 μl of 2% uranyl formate. Excess stain was immediately removed from the grids using Whatman No. 1 filter paper, and an additional 3 μl of 2% uranyl formate was added for 20 seconds. Grids were imaged using a field-emission FEI Tecnai F20 electron microscope operated at 200 kV and equipped with an Orius charge-coupled device (CCD) camera (Gatan Inc). Biolayer Interferometry. Binding kinetics measurements were performed using an Octet RED96 BLI system (Pall ForteBio) in PBS pH 7.4, 0.01% BSA and 0.002% Tween®. Unique His-tagged ligands for each Multabody component and Fc receptor were selected and loaded onto Ni-NTA biosensors to reach a signal response of 0.8 nm. Association kinetics were measured by transferring the loaded biosensor into wells containing serial dilutions of Multabody (10–5–2.5–1.25–0.65–0.32 nM) or IgG (500–250–125–62.5–31.2–15.6 nM). Dissociation kinetics were measured by immersing the biosensor into wells containing buffer. The duration of each of these two steps was 180 s. To achieve selective binding to PGDM1400, the D368R mutation in the CD4bs of the BG5050 SOSIP.664 trimer was introduced, resulting in the inhibition of N49P7 binding to this antigen. Similarly, 93TH057, a gp120 subunit in complex with β2-microglobulin, soluble CD4 and hFcRn were produced as ligands for N49P7, iMab and Fc, respectively. Binding to 10E8 was tested using a His-tagged MPER peptide (HHHHHHNEQELLELDKWASLWNWFNITNWLWYIKKKK (SEQ ID NO: 47), purchased from GenScript). Recombinantly expressed hFcγRI and hFcγRIIa were used to measure the binding affinity of IgG and Multabody with effector function silencing mutations. Purification of BG5050 SOSIP.664 D368R, CD4, 93TH057, hFcRn, hFcγRI, and hFcγRIIa used Ni-NTA purification followed by size exclusion chromatography in 20 mM phosphate, pH 8.0, 150 mM NaCl buffer. Size-exclusion chromatography with multi-angle light scattering (SEC-MALS). A MiniDAWN TREOS and Optilab T-rEX refractometer (Wyatt) were used with an Agilent Technologies 1260 infinity II HPLC. 50 μg of 24-mer PGDM1400 scFab-ferritin fusion, T-01 MB and T-02 MB were loaded onto a Superose 6 10 / 300 (GE Healthcare) column in 20 mM sodium phosphate, pH 8.0, 150 mM NaCl. Data collection and analysis were performed using ASTRA software (Wyatt). Stability measurements. Melting temperatures (T m ) and agglomeration temperature (T agg ) was determined using the UNit system (Unchained Labs). m was obtained by measuring the centroid mean (BCM) fluorescence, whereas T agg was determined as the temperature at which a 50% increase in static light scattering at a wavelength of 266 nm was observed compared to baseline. Samples were concentrated to 1.0 mg / mL and subjected to a thermal gradient from 25° C. to 95° C. in 1° C. increments. The mean and standard error of three independent measurements were calculated using UNit analysis software. Stability under accelerated stress conditions was further analyzed. Multabodies were concentrated to 10 mg / mL, diluted in 20 mM sodium phosphate (pH 8.0), 150 mM NaCl, and incubated at 40°C for 4 weeks. Each week, the percentage of properly folded protein was calculated based on the soluble content from SEC. Samples from before (week 0) and after (week 4) the incubation period were evaluated in a PsV neutralization assay to compare the functional activity of the molecules. Virus production and TZM-bl neutralization assay. As previously described, HIV-1 subtype B backbone NL4-3.Luc.R -A panel of 14 HIV-1 quasitypes was generated by cotransfection of 293T cells with E plasmid (AIDS Research and Reference Reagent Program (ARRRP)) and a plasmid encoding a full-length Env clone (45). HIV isolates X2088.c09, ZM106.9, and 3817.v2.c59 were kindly provided by the Collaboration for AIDS Vaccine Discovery (CAVD), and pCNE8, 1632_S2_B10, THRO4156.18, 278-50, ZM197M.PB7, SF162, t257-31, Du422.1, and BG505 from the NIH ARRRP. The mutation T332N in the BG505 Env expression vector was introduced by site-directed mutagenesis using the KOD-Plus mutagenesis kit (Toyobo, Osaka, Japan). An expanded 25 HIV-1 pseudotype panel was generated by spiking HIV isolates p1054.TC4.1499, 6535, ZM214M.PL15, AC10.29, p16845, P6244_13.B5.4576, pM246F_C1G, TRJO4551, QH0692, and pCAAN5342 obtained from the NIH ARRRP. Neutralization was determined in single cycle neutralization assays using a standard TZM-bl neutralization assay (45). Briefly, IgG and multibodies were incubated with 10–15% tissue culture infectious dose of pseudovirus for 1 h at 37°C, followed by incubation in TZM-bl cells for 44–72 h. Viral neutralization was monitored by adding Britelite Plus reagent (PerkinElmer) to the cells and measuring luminescence in relative light units (RLU) using a Synergy Neo2 multimode assay microplate reader (Biotek Instruments).HIV-1 Env pseudoviruses in an extended multiclade panel of 118 PsVs were generated by transfection of Env expression plasmids with the full-length Env-deleted HIV genome SG3dEnv in 293T cells.HIV-1 pseudoviruses were incubated with Multabody (initial concentration of 10 μg / ml and titrated 6-fold, 7 times) for 1 h at 37°C before adding TZM-bl cells. Luciferase expression was quantified 48 h after infection by lysing cells and adding luciferin substrate (Promega). For neutralization assays performed with parental IgG, historical data from the Center for Virology and Vaccine Research, Harvard Medical School were used (initial concentration 50 μg / ml, titrated 5-fold, 7 times). Antibody breadth was determined using a cut-off limit of 10 μg / mL. Antibody-dependent phagocytosis. 5 μL of red fluorescent neutravidin microspheres (Invitrogen, F8775) were washed twice with PBS+0.1% BSA and incubated with 10 μg of biotinylated 93TH057 antigen. Biotinylation was performed using the EZ-link Sulfo-NHS Biotinylation Kit (Thermo Scientific, 2143) according to the manufacturer's instructions. The final volume was brought to 200 μL with PBS / 0.1% BSA and incubated overnight at 4°C with rotation. Beads were washed twice before use to remove unbound protein and resuspended in 200 μL per 5 μL of unlabeled bead volume. Immune complexes were formed by incubating 10 μL of 1, 5, and 10 μg of multibody or antibody preparations with 93TH057-coated fluorescent beads (10 μL per sample) for 2 h at 37°C. THP-1 cells (ATCC TIB-202) were cultured at 5 × 10 5 Maintain cells / mL below 5 × 10 cells / mL before incubating for 16 h at 37 °C, 5% CO2. 4Cells were added to the immune complex at a concentration of 1000 cells / well (in 200 μL). After incubation, cells were pelleted and washed with PBS before staining with Live Dead Fixable Violet stain (Invitrogen, L34995) according to the manufacturer's protocol. Cells were washed with PBS and fixed with 2% paraformaldehyde for 20 min at room temperature. Fixed cells were pelleted, washed once with FACS buffer (PBS + 10% FBS, 0.5 mM EDTA) and analyzed on an LSRII flow cytometer (BD Biosciences). Data were analyzed on FlowJo (BD Biosciences, Ashland, OR) and phagocytosis was quantified as the increase in the percentage of phagocytosis compared to 93TH057-coated beads in the absence of antibody. Anti-human FcR binding inhibitor antibody (Invitrogen, 14-9161-73) was added to the indicated samples at the recommended concentration as an additional control. PBMC infection. Peripheral blood mononuclear cells (PBMCs) were obtained from three healthy blood donors, and all donors provided written informed consent. The study was approved by the University of Toronto Research Ethics Board (protocol #00037384). Blood was collected into heparinized vacutainers (BD Biosciences), and PBMCs were subsequently isolated using density centrifugation with Lymphoprep (StemCell Technologies, catalog number 07861). PBMCs were activated with phytohemagglutinin (PHA; Gibco) in the presence of recombinant human IL-2 (50 U / mL) in complete RPMI medium (Wisent) containing 10% fetal bovine serum (FBS, Wisent), 100 μg / mL streptomycin, and 100 U / mL penicillin for 72 h prior to HIV-1 infection. After 3 days of activation, 2 × 10 cells were cultured per well in RPMI + 10% FBS + 25 U / mL IL-2 by adding CXCR4-tropic laboratory isolate IIIB (150 pg p24 Gag antigen per well). 5HIV-1 infection of cells was performed on triplicate cultures of activated PBMCs in round-bottom 96-well plates seeded with 10000 cells. Multabody (T-01 MB and T-01 MB.v2) or IgG cocktail were pre-incubated with virus for 1 hour at room temperature before overlaying cells with virus. Infected cells were cultured in the presence / absence of Multabody or antibody control at doses ranging from 0.01 to 10ug / mL as indicated. Levels of HIV-1 replication were assessed by measuring extracellular release of p24 Gag protein in cell-free culture supernatants tested at day 7 post-infection using a high-sensitivity AlphaLISA p24 detection kit (PerkinElmer, Waltham, MA) on a BioTEK Synergy plate reader according to the manufacturer's protocol. Cell viability and flow cytometry. On day 7 post-infection, cells were fixed in 2% PFA and harvested for viability testing by absolute counting by flow cytometry performed using a BD LSRFortessa (Becton Dickinson). Cell viability was determined by comparing the number of live-gated cells in Multabody or antibody treated wells with the number of cells recovered from untreated control wells. Cell viability data were analyzed using a FACSDiva. Pharmacokinetic studies. In vivo studies were performed using 6-week-old NOD / Shi-scid / IL-2Rγnull (NCG strain code 572, Charles River Laboratories) immunodeficient mice, three per group. Mice were hosted by groups of 4 / 6 individuals. Each mouse was uniquely identified. Animals were kept in ventilated cages (type II (16 × 19 × 35 cm, floor area = 500 cm) under the following controlled conditions: 2)) at 22°C, 55% humidity, 12:12 h light / dark cycle 7 am:7 pm. The study was reviewed and approved by the local ethical committee (CELEAG). In this study, we used scFabs of antibodies PGDM1400, N49P7 and 10E8v4, as well as T-01 MB, consisting of scFc fragments of IgG1 Fc i) containing no mutations, and ii) containing effector function silencing mutations L234A, L235A and P329G (LALAP), and I253A mutation. In addition, we included T-01 MB.v2, consisting of the same antibody specificity i) without Fc mutations, and ii) with half-life extension mutations in IgG1 Fc (M428L / N434S). Mice received a single subcutaneous injection of 5 mg / kg Multabody or a control sample (IgG mixture matching the Fab specificity of the Multabody) in 200 μL PBS (pH 7.5). Blood samples were collected at multiple time points and serum samples were assessed for levels of circulating antibodies by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were coated with 0.5 μg / ml of MB:BG5050 D368R SOSIP.664 trimer, gp120 subunit 93TH057 and MPER peptide, His-recognized by the trimer. 6x Plates were coated with 50 μL of each tagged antigen and circulating sample concentrations were determined using reagent-specific standard curves for IgG and Multabody. HRP-Protein A (Invitrogen) was used as the secondary molecule and chemiluminescent signals were quantified using an Epoch 2 microplate spectrophotometer with Biotek Gen5 3.03 software. result The potency of HIV-1 bNAbs can be enhanced by avidity. Apoferritin is a spherical nanocage with a hydrodynamic radius of approximately 6 nm formed by the self-oligomerization of 24 identical subunits (Figure 11A). To investigate the effect of multivalency on neutralization potency, we used the self-assembly properties of the light chain of human apoferritin to multimerize antigen-binding (Fab) fragments derived from the most potent and broad-spectrum HIV-1 bNAbs targeting different HIV-1 Env epitopes. Apoferritin subunits were genetically fused to single-chain Fabs (scFabs). The scFabs were generated with a flexible linker between the light and heavy chains to ensure correct Fab heterodimerization. Self-assembly of apoferritin promoted the multimerization of the scFabs, presenting the antibody fragments around the nanocage (Figure 11B). Different densities of multimerized Fab were achieved by co-transfection of a plasmid encoding scFab-human apoferritin with different ratios of non-genetically modified human apoferritin (Figure 11C, Figure 12). Using a panel of small HIV-1 pseudoviruses (PsVs), the ability of scFab-apoferritin fusions to block HIV-1 infection was compared to the corresponding IgG (Figure 11D). Remarkably, PGDM1400, one of the most potent anti-HIV bNAbs described to date, showed 10-40 fold higher neutralization potency when multimerized via the light chain of apoferritin compared to its conventional IgG format. bNAb 10-1074 also showed a considerable improvement in neutralization potency (4-40 fold), whereas bNAbs 10E8, N49P7, and VRC01 showed no effect or a more modest enhancement. Multibodies potently and broadly neutralize HIV-1 Given these results, we sought to expand the coverage of PGDM1400 using a previously described Multabody platform based on an apoferritin split design (41). This strategy consists of splitting the four-helical apoferritin subunit into two halves (N-ferritin and C-ferritin) and fusing their N-termini to scFabs of different specificities (Figure 13A). This approach allows for the inclusion of more Fabs on the surface of the nanocage, resulting in a final molecule with higher binding activity. In addition, this design allows for the efficient combination of three different antibody specificities as well as a crystallizable fragment (Fc), endowing the molecule with IgG-like properties such as ease of purification exploiting protein A affinity (Figure 14). Specifically, the scFab PGDM1400 was combined with the scFabs of the near-pan-neutralizing antibodies 10E8v4 (modified 10E8 with improved solubility (42)) and N49P7, as well as a single-chain construct of Fc of human IgG1 isotype (scFc) (Figure 13A). To investigate whether Multabodies could also be designed to cross-target HIV-1 Env and its primary receptor, CD4, N49P7 was replaced with ibalizumab (iMab), a CD4-directed post-attachment inhibitor that has been shown to effectively inhibit HIV-1 entry (43, 44) (Figure S15A). The resulting trispecific Multabodies, designated T-01 MB and T-02 MB, respectively, formed highly decorated homogenous particles of approximately 2.4 MDa (Figures S13B-C, S15B-C) with similar thermal stability to the corresponding IgG (Figure S16). Epitope engagement by the trispecific Multabodies was evaluated in binding kinetics experiments using epitope-specific molecules: BG505 SOSIP D368R (PGDM1400), 93TH057 gp120 / CD4 (N49P7 / iMab), and MPER peptide (10E8v4) (Figure S17). Binding to the three epitope-specific antigens with high apparent binding affinity and no detectable dissociation confirms the presence of three antibody specificities in the Multabody (FIG. 13d, FIG. 15d). The neutralization potency and breadth of the multibodies was initially evaluated against a panel of 14 PsVs in a standardized in vitro TZM-bl neutralization assay (45). The 14-PsV panel was designed to include low-sensitivity PsVs with at least one PsV resistant to each bNAb evaluated (cutoff IC 50 The IC of multibody was set at 10 μg / mL. 50 Values ​​and ranges were compared to (i) each individual IgG, (ii) an IgG cocktail containing the same relative amounts of each IgG present in the multibody, and (iii) the N6 / PGDM1400x10E8v4 trispecific antibody (46). T-01 MB and T-02 MB had median IC values ​​of 0.009 μg / mL (3.9 pM) and 0.008 μg / mL (3.5 pM), respectively. 50 The 93% and 100% widths (cutoff IC 50 was set to 10 μg / mL (Figure 13E, Figure 15E, and Table 8). Therefore, the IC of the IgG cocktail and trispecific antibodies 50 Compared with the median IC values ​​calculated in μg / mL and nM for the multibody, respectively 50 There was a decrease of approximately one and two orders of magnitude in the individual IC 50 Inspection of the values ​​revealed that PsV resistant to PGDM1400 IgG neutralization was also less sensitive to Multabody (Figure S13F, Figure S15E and Table 8). These data suggested that the neutralizing properties of Multabody are largely dependent on one of the three antibody specificities within the particle, in this case PGDM1400. [Table 8] Engineering the apoferritin scaffold To further improve the neutralization properties of the Multabody, several modifications were introduced to its design, creating a second generation version (MB.v2). In the original MB, the scFc is located at the N-terminus of the N-ferritin half, and only one Fab, either Fab2 or Fab3, is incorporated into the Multabody per each functional Fc homodimer (Figure 18A, top). In comparison, the optimized MB.v2 contains many more Fabs per Fc homodimer. To achieve this, a monomeric Fc fragment (i.e., one Fc chain) and a scFab are placed at the C-terminus and N-terminus of the C-ferritin half, respectively (Figure 18A, bottom, Figure 19A). As a result, dimerization of functional Fc homodimers drives the assembly of MB.v2 particles together with split-ferritin complementation and ferritin subunit oligomerization (Figure 18A, Figure 19B). Importantly, homodimerization to form one functional Fc ensures an organization of four Fabs (i.e., two Fab2s and two Fab3s) that is different from PGDM1400, thus favoring a more balanced binding activity for each of the three Fabs in the fully assembled MB.v2. The optimized Multabody design was tested in the T-01 background (PGDM1400, N49P7, 10E8v4) targeting three epitopes on HIV-1 Env. The resulting Multabody (T-01 MB.v2) organized into well-formed spherical particles with no significant difference in morphology compared to the previously characterized T-01 MB (Figure 18B). Antigen binding to BG505 SOSIP D368R, 93TH057 gp120, and MPER peptides confirmed the correct folding of the three Fab specificities in T-01 MB.v2 (Figure 18C). Furthermore, the new Multabody version retained the same high thermostability reported for T-01 MB, confirming the high thermostability of the T aggThe value is 67°C (Figure 18D). Multabodies were concentrated to 10 mg / mL and subjected to accelerated stability studies by incubating them at 40°C for 4 weeks. Assessment of the amount of soluble protein over time revealed that the Multabodies were highly stable under these conditions, with more than 70% of the sample remaining soluble for 30 days. Stability was further confirmed by the minimal loss of neutralizing potency observed for Multabodies at week 4 compared to week 0 potency (Figure 18E). The pharmacokinetics of multibodies are similar to the corresponding IgG. Antibody Fc domains have the ability to interact with various receptors, including the Fc gamma receptor (FcγR') and neonatal Fc receptor (FcRn), which confer effector functions and in vivo half-life, respectively. However, the binding activity of Fc can adversely affect the circulation time of molecules with multiple Fc fragments (41, 47). Indeed, T-01 MB showed strong binding to Fc receptors, including human FcRn, at physiological pH (Figures 20A-B), and high and low affinity to FcγR' (Figure 20C). Therefore, we introduced a unique combination of LALAP (L234A, L235A, and P329G) and I253A mutations in the Fc of T-01 MB to reduce binding to FcγR and FcRn, respectively, and achieve binding comparable to that observed for IgG1 molecules (Figures 20A-C). T-01 MB.v2 showed a binding profile more similar to IgG1, exhibiting comparable binding to human FcRn at acidic pH and no binding at physiological pH even in the case of the half-life extension mutation LS (M428L / N434S) (Figure 20A-B). Binding of T-01 MB.v2 to FcγR' resulted in a low binding profile similar to that obtained by the LALAP FcR-silencing mutation in T-01 MB (Figure 20C). The different binding patterns observed for the two MB versions are likely due to different arrangements of the Fc fragments within the molecule (Figure 18A, Figure 19A). Despite the low binding to FcγRI, phagocytosis experiments using antigen-coated beads showed that both Multabody formats induced Fc-dependent internalization in THP-1 cells at levels similar to those achieved with the corresponding IgG mixtures (Figure 20D). Next, the in vivo bioavailability of both Multabody formats with and without engineered Fc was tested. A single dose of 5 mg / kg was administered subcutaneously to NOD / Shi-scid / IL-2Rγnull (NCG) immunodeficient mice, and the amount of each molecule in serum was measured every 2 days for 15 consecutive days. As expected from in vitro characterization, only the Fc-engineered Multabodies with IgG-like binding profiles exhibited similar decay rates over days of in vivo exposure as the parental IgG cocktail (Figure 20E). Multabody administration was well tolerated with no weight loss (Figure 20F) or visible adverse effects. Exceptional potency and pan-neutralization breadth achieved with MB.v2 The neutralization profile of T-01 MB.v2 was evaluated against a PsV panel generated by spiking 11 HIV-1 strains highly resistant to PGDM1400 to our previous panel. The resulting 25-PsV panel included PsV mutants resistant to PGDM1400 IgG neutralization (cutoff IC 50 56% of the pan-neutralization breadth (IC at 100% viral coverage, cut-off IC set at 10 μg / mL) of the corresponding IgG cocktail when tested against an extended multi-clade panel of 118 PsVs. As expected, the breadth and potency of T-01 MB were greatly affected in the presence of PGDM1400-resistant PsVs (Figures 21A-B, Table 8). However, being engineered, the neutralization profile of antibodies N49P7 and 10E8v4 was more prevalent in T-01 MB.v2, allowing this optimized multabody to achieve pan-neutralization while maintaining the enhanced neutralization potency previously observed for this type of molecule (Figures 21A-B, Table 8). When tested against an extended multi-clade panel of 118 PsVs, T-01 MB.v2 surpassed the pan-neutralization breadth (IC at 100% viral coverage, cut-off IC set at 10 μg / mL) of the corresponding IgG cocktail. 50 ), but showed significant neutralizing activity (Figures 21C-D, 22A, and Table 9). Specifically, the IgG cocktail and T-01 MB each had an IC of 0.001 μg / mL. 50In contrast, T-01 MB.v2 was only able to neutralize 9% and 8% of PsV with IC values ​​of 0.001 μg / mL, whereas 50% of PsV was still neutralized with IC values ​​of 0.001 μg / mL. 50 Notably, the multibody neutralized the IL-1 receptor with a median IC value of only 0.0009 μg / mL (0.4 pM) (Figure 21C). 50 Thus, compared to the IgG cocktail, it achieved pan-neutralization that was 32-fold and 490-fold more potent in mass and molar concentrations, respectively ( FIG. 21D ). In addition, the IC 80 confirmed a tendency for neutralization superior to both individual IgG and IgG cocktails, with a median IC of 0.005 μg / mL (2.2 pM). 80 The IgG neutralization rate was 96% for all viral strains tested (Figures 21C-D, Figure 22A, and Table 9). Importantly, the multabody also blocked infection of primary peripheral blood mononuclear cells (PBMCs) with a replication-competent CXCR4-tropic HIV-1 IIIB strain (Figure 22B), demonstrating higher potency than a matched IgG mixture and without any effect on cell viability (Figure 22C). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] Consideration Recent AMP clinical trials have highlighted the anticipated importance of both potency and breadth of bNAbs to be promising therapeutics capable of protecting against HIV-1 infection. Leveraging the principles of antibody avidity used in previously described Multabody technology to improve antibody potency against SARS-CoV-2 (41), here we engineered a second-generation Multabody platform that offers exceptional neutralization breadth and potency against the vast sequence diversity of HIV-1. The most striking feature of the optimized Multabody design compared to the first generation Multabody format (41) is the relative number of self-associating Fabs per functional Fc domain. In contrast to the 1:1 Fc, 10E8v4 / N49P7 ratio imposed by the design of the Multabody platform mentioned above, in the T-01 MB.v2 design, two N49P7 Fabs and two 10E8v4 Fabs are incorporated into the MB per dimeric Fc. The higher the number of these two Fabs in the optimized Multabody, the more favorable their binding activity and therefore their contribution to the neutralization signature of the particle. This is in contrast to the T-01 MB, which relies primarily on the neutralization properties of PGDM1400. The more balanced contribution of each antibody is reflected in a median IC of 0.0009 μg / mL. 50 This is reflected in the better functional properties of T-01 MB.v2, which showed 100% interclade neutralization coverage with IC values ​​of <1 μg / ml. Furthermore, viral infection with 83% of the 118 pseudoviruses tested was achieved with IC values ​​of <1 μg / ml. 80 value, which has recently been proposed as the potency threshold required to confer in vivo protection in humans (35). However, it is unclear whether the predictor of protection should be considered in terms of mass or molar concentration. Indeed, despite the similar hydrodynamic radius and geometric size of Multabodies compared to IgM (41), Multabodies are approximately 10 times heavier in molar mass compared to IgG. Thus, if molar concentration is the relevant in vivo measure associated with protection, T-01 MB.v2 has a very low median IC of 0.4 pM. 50Correspondingly, IC values ​​of less than 6.7 nM (1 μg / ml molar equivalent to IgG) 80 A potency of 96% of the 118-HIV-1 PsV strain was achieved. These remarkable neutralizing properties exceed those obtained with previously described bispecific and trispecific antibodies (46, 48-50). In these antibody formats, the limited avidity precludes the combination of both high avidity and multispecificity, and therefore the potency and breadth are limited to those of the parent mAb. In the field of biological therapeutics, there is an increasing trend towards the development of molecules with high valency. Strategies range from the generation of 12-valent IgM-like molecules when the mu-tail for IgM is added to the constant region of IgG (51, 52) to the design of alternative antibody formats. Among them are the fusion of Fab in a linear head-to-tail fashion (53), appended IgG (54-56), or combinations of diabodies in tandem (Tamdab) (57) or fused to the CH3 of IgG (Didiabody) (58). Furthermore, the use of multimerizing scaffolds such as p53 (59), leucine zipper helices (60), streptavidin (61), barnase-barstar modules (62), virus-like nanoparticles (63), and more recently de novo antibody cage-forming proteins (64) have been used to overcome the limitations of IgG bivalency and improve the bioactivity of antibodies. Although attractive, these approaches face different challenges for successful development as therapeutics. Multimeric antibody formats relying on variable fragments of antibodies (Fv) are often associated with low stability and therefore high tendency to aggregate (65). Furthermore, dissociation of non-covalent bonds, described by the affinity constant of the complex, may limit the in vivo long-term stability of the molecule. In sharp contrast, Multabodies are built on complete IgG components (Fab and Fc) fused to a thermostable, functionally silent human apoferritin light chain scaffold and are therefore highly stable IgG-like molecules even under heat stress. A murine surrogate Multabody administered subcutaneously to previously immunocompetent C57BL / 6 mice showed undetectable levels of anti-drug antibodies similar to its parental IgG, providing evidence of the potentially low intrinsic immunogenicity of the Multabody platform (41). We propose that future studies in higher organisms will help determine the immunogenicity of Multabodies encoded by human-derived sequences, which may be primarily described by the properties of the underlying antibody sequences. Bioavailability of large biologics is an additional challenge related to engineered approaches to increase binding activity (63). Multabodies have been engineered to contain an Fc domain, thus allowing FcRn-mediated recycling of the molecule. As a result of Fc binding activity, the binding of T-01 MB to FcRn at pH 6.0 was improved by several orders of magnitude, but the simultaneous affinity improvement at pH 7.4 limited the application of this strategy towards half-life enhancement, and several mutations were required to reduce Fc binding to FcRn and FcγR so as not to exceed the binding affinity observed for IgG. Such enhanced Fc receptor binding activity was not observed in the case of T-01 MB.v2, where the fusion of the Fc chain at the C-terminus of apoferritin led to the formation of particles with an inverted and more distally located Fc domain, resulting in reduced Fc binding activity. Similar to previous work with mouse surrogate Multabodies (41), the Fc avidity modulation strategy successfully yielded Multabody molecules with remarkably similar decay rates over time as the parental IgG cocktail. In addition to favorable pharmacokinetic profiles, Multabodies of both formats have residual binding to FcγR-induced Fc-mediated phagocytosis to levels similar to the parental IgG mixture in vitro, at least in the THP-1 line co-expressing both FcγRI and FcγRIIa (66). Future experiments will be required to fully characterize the ability of Multabodies to induce immune effector functions and their in vivo inclusion. From the limited number of antibody specificities characterized in this study, it was observed that antibodies targeting epitopes located at the apex of the HIV-1 Env trimer, such as PGDM1400, seemed to provide the greatest benefit in neutralization potency when formulated as a Multabody. This increase in potency was less evident when the epitope was located closer to the viral membrane, as was the case for 10E8. The dependency on epitope location for potency enhancement could be further influenced by low surface spike density (67), the configuration of those sparse Env trimers on the HIV-1 surface (68), or the accessibility of certain epitopes that may be more or less sterically occluded. In light of this, it will be interesting to explore how the potency of antibodies against viruses with higher surface density and closely spaced spikes can be enhanced by the Multabody platform, as well as to further determine the impact of epitope location on the avidity-mediated potency enhancement. Replacement of N49P7 by iMab, a CD4-directed post-attachment inhibitor, resulted in a functional Multabody with potent neutralizing activity, demonstrating that cross-targeting of viral epitopes and cellular receptors can be achieved by this type of particle. This data raises the intriguing possibility that Multabody technology could also be implemented in other fields to facilitate the binding of receptors across separate entities, such as cell-cell interactions in immunotherapy. Overall, our protein engineering studies demonstrate the versatility of the human apoferritin antibody as a modular nanocage to engineer antibody avidity and multispecificity toward enhanced functionality. References 1.A.J.Conley, et al., Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody.Proc.Natl.Acad.Sci.U. S. A. 91, 3348-3352 (1994). 2.G. Stiegler, et al., A Potent Cross-Clade Neutralizing Human Monoclonal Antibody against a Novel Epitope on gp41 of Human Immunodeficiency Virus Type 1.AIDS Res.Hum.Retroviruses 17, 1757-65 (2001). 3.M. B. 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Kwon, et al., Optimization of the Solubility of HIV-1-Neutralizing Antibody 10E8 through Somatic Variation and Structure-Based Design.J.Virol.90, 5899-5914 (2016). 43.J.M. Jacobson, et al., Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody, in human immunodeficiency virus type 1-infected adults.Antimicrob.Agents Chemother.53, 450-457 (2009). 44.D. R. Kuritzkes, et al., Antiretroviral Activity of the Anti-CD4 Monoclonal Antibody TNX-355 in Patients Infected with HIV Type 1.J.Infect.Dis.189, 286-291 (2004). 45. D. C. Montefiori, Measuring HIV neutralization in a luciferase reporter gene assay. Methods Mol. Biol. 485, 395 - 405 (2009). 46. L. Xu, et al., Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques. Science 358, 85 - 90 (2017). 47. O. S. Qureshi, et al., Multivalent Fcγ - receptor engagement by a hexameric Fc - fusion protein triggers Fcγ - receptor internalisation and modulation of Fcγ - receptor functions. Sci. Rep. 7 (2017). 48. Refer to M. Asokan, et al., Bispecific Antibodies Targeting Different Epitopes on the HIV - 1 Envelope Exhibit Broad and Potent Neutralization. J. Virol. 89, 12501 - 12512 (2015). 49. S. N. Khan, et al., Targeting the HIV - 1 Spike and Coreceptor with Bi - and Trispecific Antibodies for Single - Component Broad Inhibition of Entry. J. Virol. 92, e00384 - 18 (2018). 50.J.J. Steinhardt, et al., Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity.Nat.Commun.9(2018). 51.R. I. Smith, M.J.Coloma, S. L. Morrison, Addition of a mu-tailpiece to IgG results in polymeric antibodies with enhanced effector functions including complement-mediated cytolysis by IgG4.J.Immunol.154, 2226-36 (1995). 52.T. Olafsen, B. I. Rasmussen, L. Norderhaug,O .S. Bruland, I. Sandlie, IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells.Immunotechnology 4, 141-153 (1998). 53.K. Miller, et al., Design, Construction, and In Vitro Analyses of Multivalent Antibodies.J.Immunol.170, 4854-4861 (2003). 54.C. Wu, et al., Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin.Nat.Biotechnol.25, 1290-1297 (2007). 55.C. Klein, W. Schaefer,J.T. Regula, The use of CrossMAb technology for the generation of bi- and multispecific antibodies.MAbs 8, 1010-1020 (2016). 56.A. Steinmetz, et al., CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications.MAbs 8, 867-878 (2016). 57.S. M. Kipriyanov, et al., Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.J.Mol.Biol.293, 41-56 (1999). 58.D. Lu, et al., Di-diabody:A novel tetravalent bispecific antibody molecule by design.J.Immunol.Methods 279, 219-232 (2003). 59.S. Kubetzko、E. Balic、R. Waibel、U. Zangemeister-Wittke、A. Pluckthun、PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5:Effects on tumor targeting.J.Biol.Chem.281, 35186-35201 (2006). 60.P. Pack, K. Muller, R. Zahn, A. Pluckthun, Tetravalent miniantibodies with high avidity assembling in Escherichia coli.J.Mol.Biol.246, 28-34 (1995). 61.S. M. Kipriyanov, et al., Affinity enhancement of a recombinant antibody:Formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion.Protein Eng.9, 203-211 (1996). 62.S. M. Deyev, R. Waibel, E. N. Lebedenko, A. P. Schubiger, A. Pluckthun, Design of multivalent complexes using the barnase·barstar module.Nat.Biotechnol.21, 1486-1492 (2003). 63.M. A. G. Hoffmann, et al., Nanoparticles presenting clusters of CD4 expose a universal vulnerability of HIV-1 by mimicking target cells.Proc.Natl.Acad.Sci.U. S. A. 117, 18719-18728 (2020). 64.R. Divine, et al., Designed proteins assemble antibodies into modular nanocages.Science 372, eabd99947 (2021). 65.A. Worn, A. Pluckthun, Different equilibrium stability behavior of scFv fragments:Identification, classification, and improvement by protein engineering.Biochemistry 38, 8739-8750 (1999). 66.H. B. Fleit, C. D. Kobasiuk, The Human Cell Line THP-1 Expresses Fc-gamma-RI and Fc-gamma-RII.J Leukoc Biol 49, 556-565 (1991). 67.P. Zhu, et al., Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions.Proc.Natl.Acad.Sci.U. S. A. 100, 15812-15817 (2003). 68.J.Chojnacki, et al., Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy.Science 338, 524-528 (2012). 69.M. Graille, et al., Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.Structure 9, 679-687 (2001). 70.T. Zhou, et al., Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01.Science 329, 811-817 (2010). 71.EO Freed, DJMyers, R. Risser, Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.J.Virol.63, 4670-4675 (1989). Sequence Listing Underlining within the sequences indicates linker sequences, bold text within the sequences indicates ferritin or ferritin subunit sequences, and boxed and bolded residues indicate residues that are mutated relative to the reference molecule, e.g., IgG1 Fc. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0220] Equivalents / Alternative Embodiments While the invention has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover generally any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including such departures from the present disclosure that are within known or customary practice in the art to which this invention pertains, and may be applied to the essential features of the invention as described hereinabove.

Claims

1. (a) a plurality of first fusion polypeptides, each first fusion polypeptide comprising: (1) an Fc polypeptide; (2) said Fc polypeptide linked to a nanocage monomer or a subunit thereof, said Fc polypeptide comprising an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class; (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising: (1) an antigen-binding antibody fragment; and (2) the antigen-binding antibody fragment linked to a nanocage monomer or subunit thereof; (1) when the Fc polypeptide is an IgG1 Fc polypeptide, the antigen-binding fragment is not a Fab fragment that binds to SARS-CoV-2, and / or (2) when the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds to CD19.

2. The self-assembled polypeptide complex of claim 1 , wherein the nanocage monomer is a ferritin monomer.

3. The self-assembled polypeptide complex of claim 2 , wherein the ferritin monomer is a ferritin light chain.

4. The self-assembled polypeptide complex of claim 3, which does not contain any ferritin heavy chain or ferritin heavy chain subunit.

5. The self-assembled polypeptide complex of claim 3 or 4, wherein the ferritin monomer is human ferritin.

6. The self-assembling polypeptide complex of claim 1 , wherein the Fc polypeptide is an IgG1 Fc polypeptide.

7. The self-assembling polypeptide complex of claim 1 , wherein the Fc polypeptide is an IgG2 Fc polypeptide.

8. The self-assembling polypeptide complex of claim 1 , wherein the antigen-binding antibody fragment comprises a light chain variable domain and a heavy chain variable domain.

9. The self-assembled polypeptide complex of claim 1 , wherein the one or more mutations comprise a mutation or set of mutations associated with altered binding to FcRn.

10. 10. The self-assembled polypeptide complex of claim 9, wherein the mutation or set of mutations comprises mutations at one or more of the following residues: M252, I253, S254, T256, K288, M428, and N434 (numbering according to the EU index), or a combination thereof.

11. 11. The self-assembling polypeptide complex of claim 10, wherein the mutation or set of mutations comprises mutations at M428 and N434 (numbering according to the EU index).

12. 12. The self-assembled polypeptide complex of claim 11, wherein the mutation or set of mutations comprises M428L and N434S (numbering according to the EU index) mutations.

13. The self-assembled polypeptide complex of claim 9 or 10, wherein the altered binding to FcRn is decreased binding to FcRn.

14. 11. The self-assembled polypeptide complex of claim 10, wherein the mutation or set of mutations associated with reduced binding to FcRn is selected from the group consisting of I253A, I253V, and K288A (numbering according to the EU index), and combinations thereof.

15. The self-assembled polypeptide complex of claim 1 , wherein the one or more mutations comprise a mutation or set of mutations associated with an altered effector function.

16. 16. The self-assembled polypeptide complex of claim 15, wherein the Fc polypeptide is an IgG1 Fc polypeptide and the mutation or set of mutations comprises mutations at one or more of the following residues: L234, L235, G236, G237, P329, and A330 (numbering according to the EU index), or a combination thereof.

17. The self-assembled polypeptide complex of claim 15 , wherein the altered effector function is a reduced effector function.

18. 18. The self-assembled polypeptide complex of claim 17, wherein the mutation or set of mutations associated with reduced effector function is selected from the group consisting of LALA (L234A / L235A), LALAP (L234A / L235A / P329G), G236R, G237A, and A330L (numbering according to the EU index).

19. the nanocage monomer or subunit thereof is a ferritin monomer subunit; a. each first fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, or b. The self-assembling polypeptide of claim 1, wherein each first fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin.

20. The self-assembled polypeptide complex of claim 1, characterized in that after administration of a composition comprising the self-assembled polypeptide complex, the self-assembled polypeptide complex has a half-life substantially similar to the half-life of a reference IgG molecule administered by the same route of administration and in a similar composition.

21. The self-assembling polypeptide complex of claim 1, wherein the self-assembling polypeptide complex induces antibody-dependent cellular phagocytosis (ADCP) in an in vitro model of ADCP.

22. The self-assembled polypeptide complex of claim 21, wherein the ADCP is induced at a level of internalization of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the target.

23. (1) an Fc polypeptide; (2) a fusion polypeptide comprising said Fc polypeptide linked to a nanocage monomer or subunit thereof, wherein said Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, wherein said one or more mutations comprise a mutation or set of mutations associated with altered binding to FcRn and / or altered effector function; (1) when the Fc polypeptide is an IgG1 Fc polypeptide, the antigen-binding fragment is not a Fab fragment that binds to SARS-CoV-2, and / or (2) when the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds to CD19.

24. the nanocage monomer or subunit thereof is a ferritin monomer subunit; a. each first fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin, or b. The fusion polypeptide of claim 23, wherein each first fusion polypeptide comprises a ferritin monomer subunit that is an N-half ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is a C-half ferritin.

25. (1) An Fc polypeptide; (2) a fusion polypeptide comprising the Fc polypeptide linked to a nanocage monomer or a subunit thereof, wherein the Fc polypeptide comprises an Fc chain comprising one or more mutations including a mutation at K288, a mutation at I253V, or a combination thereof, wherein the numbering is according to the EU index.

26. 26. The fusion polypeptide of claim 25, wherein the one or more mutations include a I253V mutation.

27. 26. The fusion polypeptide of claim 25, wherein the one or more mutations include a K288A mutation.

28. The one or more mutations are selected from the following set of mutations: (a) K288A / P329G; or (b) L234A / L235A / P329G (LALAP) / K288A 28. The fusion polypeptide of claim 27, comprising one of:

29. The fusion polypeptide of any one of claims 25 to 28, wherein the nanocage monomer is a ferritin monomer or a ferritin monomer subunit.

30. A self-assembled polypeptide complex comprising the fusion polypeptide of any one of claims 25 to 28.

31. A composition comprising the self-assembling polypeptide complex of claim 1 and a pharmaceutically acceptable excipient.

32. 32. The composition of claim 31 for use in the treatment of a disease or condition.

33. 33. The composition for use of claim 32, wherein the disease or condition is an infectious disease, cancer, or an autoimmune disease.

34. 32. Use of the composition of claim 31 for the manufacture of a medicament for treating a disease or condition.

35. 35. The use of claim 34, wherein the disease or condition is an infectious disease, cancer, or an autoimmune disease.

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